JPH0835155A - Material for reinforcing microorganism-immobilizing carrier - Google Patents

Abstract

PURPOSE:To obtain the reinforcing material used for the microorganism- immobilizing carrier and comprising the bacterium cellulose of ribbon-like microfibrils and added to a carrier for immobilizing microorganisms to impart durability against stress destruction and deformation and a cell leakage- preventing property on usage to the carrier. CONSTITUTION:A microorganism producing bacterium cellulose, such as Acetobacter aceti subspecies xylinum ATCC 10821, is cultured to form a gel-like membrane containing a bacterium cellulosic polysaccharide. The membrane is disintegrated and filtered with a pulp-disintegrating machine to obtain the paste of the bacterium cellulose comprising ribbon-like microfibrils. A gelatin used as the carrier of a microorganism is mixed with the above-described disintegration product, homogeneously dispersed and mixed with the microorganism, further mixed with transglutaminase, and subsequently subjected to the gelation of the gelatin to obtain the microorganism-immobilized gelatin gel reinforced with the bacterium cellulose.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a molding material having high elasticity and high strength, which is excellent in tensile strength, stretch resistance and is obtained by containing a specific cellulose produced by bacteria.

This molding material can be used not only as paper and other various sheets, but also as thread-like or various three-dimensional molded articles.

[0003]

2. Description of the Related Art Conventionally, as cellulose produced by bacteria, Acetobacter xylinum (Acetob)
It is known to use a sheet-shaped product produced by ACTC xylinum) ATCC 23769 for a medical pad (Japanese Patent Laid-Open No. 59-120159).

On the other hand, various conventional molding materials are known, and as for cellulose, fibers used in thread form, sheet form, various three-dimensional molded products, cellophane obtained by once dissolving and processing a cellulose derivative, There are celluloid etc. In addition, various synthetic polymer materials have been developed, and among them, there is one in which molecular chains are arranged in a certain direction to particularly increase the mechanical strength in that direction.

[0005]

The conventional mechanical strength of various plant-derived celluloses and cellulose derivatives is not so large, for example, the elastic modulus of sheet-shaped celluloid and cellophane is about 2-3 GPa at most.

[0006] Among synthetic polymer materials, some of which have molecular chains arranged in a certain direction have elastic moduli equivalent to those of metals and inorganic materials in one direction, but have low elastic moduli in other directions. Therefore, the use was originally limited to use as a high-strength material. For this reason, there is a demand for a structural material having excellent anisotropy in the arrangement of the molecules and excellent in strength, but the elastic modulus is low in a polymer substance in which the molecules are randomly arranged. Polyester film, aramid sheet, polyimide film, etc. are known as high-performance molding materials made of synthetic polymers.
The elastic modulus was at most about 4 to 7 GPa.

Although the above-mentioned examples use cellulose produced by bacteria, its use is limited to medical pads, and it is completely known that it has high utility value as a material in the field of high mechanical strength. Didn't.

An object of the present invention is to provide a molding material having high elasticity and high strength, which is superior to the conventional molding materials in tensile strength and stretch resistance.

Another object of the present invention is to provide a molding material which is excellent in hydrophilicity in addition to the high mechanical strength and has no toxicity problem.

Still another object of the present invention is an enzyme having a function of improving strength, preventing stickiness, and preventing leakage of bacterial cells,
It is to provide a high-strength molding material that can be used as an immobilization carrier for microorganisms and the like.

Yet another object of the present invention is to impart high conductivity in various fields by imparting conductivity, magnetism, high insulation, thermal conductivity, weather resistance, chemical resistance and the like in addition to high mechanical strength. Providing mechanical strength materials.

[0012]

Means for Solving the Problems The inventors of the present invention have conducted various studies to achieve these objects, and the cellulose composed of ribbon-shaped microfibrils produced by microorganisms has extremely large mechanical strength such as tensile strength, It was found that the above object can be achieved by using a material containing this bacterial cellulose as a molding material, and the present invention has been completed based on this finding.

That is, the present invention relates to a high mechanical strength molding material containing bacterial cellulose composed of ribbon-shaped microfibrils.

As shown in the electron micrograph of FIG. 1, bacterial cellulose is composed of ribbon-shaped microfibrils having a width of 100 to 500Å and a thickness of 10 to 200Å. Generally, it is obtained in the form of gel and has a water content of 9
It is 5% (w / v) or more.

This cellulose is easily decomposed by cellulase to produce glucose. That is, a cellulase (EC) was added to a 0.1% (w / v) suspension of the present cellulose.
3,2,1,4) (manufactured by Amano Pharmaceutical Co., Ltd.) was dissolved to 0.5% (w / v), and the solution was dissolved in 0.1 M acetate buffer at 30 ° C. for 24 hours.
Allowed to react for hours. As a result, it was observed that part of this substance was decomposed, and when the supernatant was developed by paper chromatography, small amounts of cellobiose, cellotriose and other cellooligosaccharides were detected in addition to glucose. In addition to this, small amounts of fructose, mannose, etc. were sometimes detected.

That is, the bacterial cellulose of the present invention includes cellulose and a heteropolysaccharide having cellulose as a main chain and glucans such as β-1,3, β-1,2. In the case of the heteropolysaccharide, the constituent components other than cellulose are hexoses such as mannose, fructose, galactose, xylose, arabinose, rhamnose and glucuronic acid, pentose sugars and organic acids. These polysaccharides may be a single substance, or two or more polysaccharides may be mixed due to hydrogen bonding or the like.

Any bacterial cellulose can be used as long as it is as described above.

Microorganisms producing such bacterial cellulose are not particularly limited, but Acetobacter aceti subsp. Xylinum (Acetobacter)
eraceti subsp xylinum) ATCC
10821 or the same Pastourian (A. pas
teurianus), the same license (A. rance)
ns), Sarcina ve
ntriculi), Bacterium xyloides (B
It is possible to use bacteria that produce bacterial cellulose, such as bacteria of the genus Pseudomonas, Pseudomonas, and bacteria of the genus Agrobacterium.

The method for culturing these microorganisms to produce and accumulate bacterial cellulose may be a general method for culturing bacteria. That is, a carbon source, a nitrogen source, an inorganic salt, and other amino acids as necessary, an ordinary nutrient medium containing organic micronutrients such as vitamins is inoculated with a microorganism,
Stir or gently ventilate. As the carbon source, glucose, sucrose, maltose, starch hydrolyzate, molasses, and the like are used, and ethanol, acetic acid, citric acid, and the like can be used alone or in combination with the above-mentioned sugars. As the nitrogen source, ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, nitrates, organic or inorganic nitrogen sources such as urea and peptone are used. As the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like are used. As organic micronutrients, amino acids, vitamins, fatty acids, nucleic acids, and peptones, casamino acids, yeast extracts, and soybean protein hydrolysates containing these nutrients are used, and auxotrophic mutations that require amino acids for growth are used. When using strains, it is necessary to further supplement the required nutrients.

The culture conditions may be normal, and the pH is 5 to 9
Then, while controlling the temperature to 20 to 40 ° C, 1 to 3
If cultivated for 0 days, bacterial cellulose accumulates on the surface in the form of a gel.

The bacterial cellulose used in the present invention may be a purified product isolated from a culture of a microorganism, or may contain impurities to some extent depending on the use. For example, residual sugar, salts, yeast extract, etc. in the culture solution may remain in the microbial cellulose. In addition, the cells may be contained to some extent.

The gel is taken out and washed with water if necessary. A chemical such as a bactericide or a pretreatment agent can be added to the washing water according to the purpose.

After washing with water, it is dried or kneaded with another kneaded product and dried before use. The drying method may be any method, but it goes without saying that it is usually necessary to perform it in a temperature range where cellulose is not decomposed. In addition, since the cellulosic material is composed of fine fibers having a large number of hydroxyl groups on the surface, the fibers may lose their fibrous form due to mutual adhesion during drying. Therefore, when it is desired to prevent this and use the fine fibrous form, it is preferable to use a method such as freeze-drying or critical point drying.

Bacterial cellulose preferably has a structure in which microfibrils are entangled with each other in order to enhance mechanical strength such as tensile strength. For this purpose, for example, a gel taken out from a culture is pressed at a right angle to be squeezed. It is effective to remove most of the free water before drying. A suitable pressing pressure is about 1 to 10 kg / cm ² . By this pressing, the dried cellulose becomes oriented according to the pressing direction. By performing an operation of stretching in one direction while applying pressure, that is, a rolling operation, the dried cellulose has an orientation in the rolling direction in addition to the pressing direction. The pressing device can be appropriately selected and used from commercially available models.

On the other hand, once disaggregating the bacterial cellulose is also effective in increasing the mechanical strength. The disaggregation may be carried out by utilizing mechanical shearing force, and can be easily disaggregated by, for example, a rotary disintegrator or a mixer. It is also effective to perform the above-mentioned pressing after disaggregation.

The high mechanical strength molding material of the present invention can be molded into various shapes such as a sheet shape, a thread shape, a cloth shape and a three-dimensional shape.

In the case of a sheet form, bacterial cellulose may be disintegrated if necessary, then layered, and if necessary pressed and dried. In addition to pressing, a sheet having a plane orientation can be obtained, and a sheet having a plane orientation and a further uniaxial orientation can be obtained by applying rolling.

It is desirable to dry the sheet which has been disintegrated and / or squeezed, by fixing it on a suitable support. By fixing to this support, the degree of plane orientation is further increased, and a sheet having high mechanical strength can be obtained. As the support, for example, a plate having a mesh structure, a glass plate, a metal plate or the like can be used. The drying temperature may be within a range where cellulose is not decomposed, and a freeze-drying method can be used in addition to the heat-drying method.

The sheet thus obtained has a structure in which microfibrils are randomly entangled with each other, as shown in FIG. Then, according to the X-ray diffraction image, the compressed product has a plane orientation, and the rolled product has a plane orientation as well as a uniaxial orientation. The elastic modulus of the sheet is usually 1
It is about 0 to 20 GPa.

The thickness of the sheet is determined depending on the application, but is usually about 1 to 500 μm.

Various additives can be added to the sheet. For example, by adding a solution of various polymeric materials (aqueous or non-aqueous), emulsion, dispersion, powder, melt, etc., depending on the characteristics of the additive, strength, weather resistance, chemical resistance, Some of water resistance, water repellency, antistatic property, etc. can be imparted. Aluminum, copper,
If a metal such as iron or zinc or carbon is added in the form of powder or thread, the electrical conductivity and thermal conductivity can be improved.
In addition, if an inorganic material such as titanium oxide, iron oxide, calcium carbonate, kaolin, bentonite, zeolite, mica, and alumina is added, heat resistance, insulation, etc. are improved depending on the type, or surface smoothness is imparted. be able to. Strength can be further increased by the addition of low molecular weight organics or adhesives. It may be colored with a dye such as phthalocyanine, an azo compound, eye, or safflower. Various other paints, dyes and pigments can be used for coloring. It can also be used as a medical sheet by adding pharmaceuticals and bactericides.

These kneaded products and additives are added in an appropriate amount so that the desired physical properties can be obtained at 97% or less. The timing of addition of these is not critical, and they may be added to the bacterial cellulose gel or the disaggregated product thereof, added after squeezing, or added after drying. In some cases, it may be added in the medium or to the culture. The addition method may be mixing or impregnation.

Layers of other materials can be laminated to such sheets. The laminate is appropriately selected depending on the purpose of use of the sheet. It can be selected from the above-mentioned kneaded materials or additives, and for example, various polymer materials can be coated to impart water resistance.

When used as paper, the bacterial cellulose gel may be disintegrated and then paper-made and dried, whereby it is excellent in tensile strength, stretch resistance and the like, and is chemically stable and has excellent water absorption and air permeability. It is possible to obtain high elasticity and high strength paper. In this case, the usual additives and treating agents used for papermaking can be used, or the kneaded products and additives described above can be selected and added.

In recent years, the demand for electrically insulating paper, heat-resistant paper, flame-retardant paper, etc. has increased, and synthetic paper, non-organic paper and the like using non-cellulosic fibers have come to be produced. When these are to be produced by the wet method, non-cellulosic fibers do not undergo hydrogen bonding, so unless pulped fibers such as fibers of polyethylene, polypropylene, polyacryconitrile, aromatic polyamide, etc. are obtained. Therefore, it is necessary to add cellulose pulp to perform papermaking. In this case, it is required to reduce the amount of pulp as much as possible to improve the insulation, heat resistance and flame retardancy. However, when blending a commonly used wood pulp, the addition amount is 20 to
It reaches 50%, and the purpose cannot be fully achieved. However,
By using bacterial cellulose instead of wood pulp, the amount of cellulose could be greatly reduced, and a paper excellent in insulation, heat resistance and flame retardancy could be obtained.
Therefore, the high mechanical strength molding material of the present invention is also effective for these.

Further, photocrosslinkable polyvinyl alcohol is said to have a better affinity for living organisms than conventional photocrosslinkable resins, and it is thought that the use of immobilizing agents such as enzymes and microorganisms will be further improved by this. ing. Also, the photoresist used when making the original plate for printing is
The resin is applied on the substrate, the pattern to be printed is projected on it to cause photo-crosslinking to cure the resin, and the uncured resin is washed away to produce the printing original plate, which is water-soluble. The photocrosslinkable polyvinyl alcohol has the advantage that it is cheaper and easier to wash than photoresists of conventional oil-soluble ones, and is expected to be applied. But,
In these cases, there has been a problem that the photocrosslinkable polyvinyl alcohol swells with water and the crosslinked structure is destroyed. However, this swelling can be prevented by adding bacterial cellulose.

In the case of forming a thread, for example, a bacterial cellulose gel or a disaggregated product thereof is washed and dried, and then dissolved in a dimethylacetamide / lithium chloride solvent or the like, and the dissolved product is water or alcohols, ketones, dioxane, Spinning may be performed using a coagulating liquid in which cellulose such as tetrahydrofuran is insoluble and a solvent of cellulose is soluble.

In the case of making a cloth, the yarn may be woven by a conventional method.

In the case of making a three-dimensional object, various plastics are kneaded with bacterial cellulose or further laminated to obtain a desired molded article. This can be substituted for various FRP products or carbon fiber products, for example.

As the immobilizing carrier, agar, carrageenan alginic acid, gelatin, collagen, polyamino acid, photocrosslinkable resin, polyacrylamide and various resins are conventionally used. Polyvinylidene chloride, polyester or the like net or fiber is used as a reinforcing material. As a result, it has been attempted to improve the strength and spin the adhesive by mixing it with these carriers. However, in this immobilization product, the reinforcing material enlarges the carrier to reduce the surface area per unit area, inhibits the diffusion of the substrate in the carrier, or the enzyme immobilized on the carrier, the biological substance, the catalyst, other There has been a problem that the concentration of the reactive substance (hereinafter referred to as “active ingredient”) is relatively decreased, and the reaction rate and the reaction yield are decreased. In addition, since the active ingredient leaks out from the immobilized carrier, there is a problem that the activity gradually decreases when used for a long period of time or repeatedly, and even if a conventional reinforcing material such as polyvinylidene chloride or polyester is added, the active ingredient may be added. It was difficult to prevent the leakage of the above from the carrier. Of the conventional immobilization carriers, a very weak gel, for example, a 6% aqueous solution of photo-crosslinkable polyvinyl alcohol cross-linked, when used as an immobilization carrier, it is fixed because water causes swelling. There is also a problem that the carrier is destroyed.

Instead of the conventional immobilizing carrier reinforcing material, bacterial cellulose can be used as it is, in a disintegrated state, or by exposing and drying these materials to use.
By solving the above-mentioned problems, it is possible to obtain an immobilization carrier having a high strength which has never been obtained.

There are the following methods for producing such an immobilized carrier. Basically, the bacterial cellulose and the following immobilized carrier material are mixed and then gelled,
It may be polymerized or molded. If necessary, the active ingredient for the purpose of immobilization may be added together for immobilization during the mixing, or the active ingredient may be immobilized after the immobilization carrier is manufactured.

The carrier material is not particularly limited as long as it can be mixed with the bacterial cellulose, but the following materials can be used, for example. Agarose, dextran, cellulose, cellulose derivatives, alginic acid,
Alginate, chitin, chitosan, collagen, albumin, amino acid polymer, polystyrene, polyacrylamide, tannin, silicone rubber, casein, agar, carrageenan, polyurethane, poly-2-hydroxyethylmethacrylic acid, polyvinyl chloride, γ-methylpolyglutamic acid, Polyvinylpyrrolidone, polydimethylacrylamide, photo-crosslinkable resin, polyethylene glycol derivative, polypropylene glycol derivative, polybutadiene derivative, collodion, nylon, polyurea, silica gel, silicon derivative, phenylsiloxane, fibrin, cellulose nitrate, hydrocarbon, phospholipid, calcium phosphate gel , Phenoxyacetylated products, glucomannan, etc.

The method for producing the immobilization carrier will be described below.
When the bacterial cellulose is in a gel form as it is produced by microorganisms, it is a dried product obtained by drying the gel form, or a dry product obtained by disintegrating and drying the gel form. If the material does not maintain the fibrous form, the carrier material is made into a solution or melted with an appropriate solvent so as to have fluidity, and then impregnated into the bacterial cellulose. An immobilization carrier is obtained by gelling. The method of gelation varies depending on the carrier material. For example, in the case of sodium alginate, it may be mixed and then added in a calcium chloride solution, and in the case of agar, the temperature may be lowered.

When the bacterial cellulose is in a disaggregated state, or in a dried state in which a fibrous form obtained by a method such as freeze-drying or critical point drying is left, the bacterial cellulose and the carrier material are used. Differently from the above-mentioned impregnation, the immobilized carrier is obtained by gelling, polymerizing or molding after mixing by an ordinary method.

The concentration of the bacterial cellulose in the immobilization carrier is 0.01% to 99%, preferably about 0.1 to 2%.

The shape of the immobilization carrier obtained by the above method can be freely selected according to the type and method of the reaction. For example, when it is packed in a column or put in a stirring tank, it may be processed into a ball shape or may be formed into a rod shape or a film shape if necessary. When the immobilized carrier is produced by mixing the disaggregated bacterial cellulose with a carrier material and then gelling, polymerizing or molding the immobilized carrier, the immobilized carrier may be processed and molded in the same manner as in the conventional method.

On the other hand, in the case of directly producing the immobilized carrier from the gel form as produced by the microorganism, the carrier material may be impregnated in the gel cellulose gel and then gelled. In order to process it into a predetermined shape, the gel-like bacterial cellulose may be processed into a required shape and then impregnated with a carrier material, while the immobilized carrier may be manufactured and then processed into a required shape. good.

The active ingredient immobilized on the immobilization carrier of the present invention is an enzyme, a microorganism, a substance derived from a living body, a catalyst or other reactive substance, and any substance generally used may be used. Examples of active ingredient enzymes include aspartase, L-aspartate β-decarboxylase, L-leucine dehydrogenase, hydantoinase, DL-2-amino-Δ.
² -thiazoline-4-carboxylic acid hydrolase, dihydropyrimidinase, acylase acting on acylamino acid,
Tryptophanase, tryptophan synthetase, tyrosinase, fumarase, histidine ammonia lyase, L-amino acid oxidase, α-amylase, β-
Amylase, invertase, glucose isomerase, penicillin acylase, cephalosporin acylase, 5'-AMP deaminase, arginine deiminase, aldolase, cysteine desulfhydrase, methioninase, phosphodiesterase, chymotrypsin, trypsin, papain, naringinase, lactase, glucoamylase, Leucine aminopeptidase,
Pepsin, aminolactam hydrolase, amylolactam racemase, glutamate dehydrogenase, aspartate decarboxylase, pullulanase, hyaluronidase, 1,4-α-glucan phosphorylase, cyclodextrin glycosyltransferase, dextran sucrase, α-glucosidase, β-glucosidase, hexokinase, Δ ¹ -dehydrogenase, 11β
-Hydroxylase, 20β-dehydrogenase, 3β-dehydrogenase, Δ ¹ -hydroxylase, steroid esterase,
5'phosphodiesterase, ATP deaminase, acetate kinase, adenylate kinase, adenosine kinase,
Carbamyl phosphokinase, pyruvate kinase, glycolytic enzyme chymosin, alkaline protease, rennin, pronase, protease, catalase, lysozyme, D
-Oxynitrase, alcohol dehydrogenase, glucose-6-phosphate dehydrogenase, nitrate reductase, nitrite reductase, rhodanese, glutaminase, uricase, peroxidase, lipase, cholesterol oxidase, penicillinase, alkaline phosphatase, acid phosphatase, acetylcholinesterase, and the like. To be Examples of microorganisms of the active ingredient include Brevibacterium ammoniagenes, Escherichia coli, Erviherbicola, Streptococcus faecalis, Pseudomonas putida, Serratia.
Macerans, Bacillus subtilis, Acetobacter
Aceti, Lactobacillus delbrettii, Pseudomonas fluorescens, Micrococcus luteus,
Bacillus megaterium, Penicillium chlorigenum, Candida tropicalis, Saccharomyces cerevisiae, etc. are mentioned. In addition, animal cells, plant cells, etc. may be immobilized if necessary.

The immobilized product thus obtained can be used for the production of useful substances such as medicines and foods using microorganisms and enzymes. It can also be used as a bioreactor in analytical and industrial production processes.

The catalyst and other reactive substances may be organic or inorganic substances that can coexist with the bacterial cellulose and are used in ordinary chemical reactions. These materials can be packed in a column and used as a reaction bed, or can be used for various chromatography. On the other hand, the immobilized carrier can be used for physically separating and selecting or purifying the target substance by selecting the particle size and size.

[0052]

[Function] Bacterial cellulose is composed of ribbon-shaped microfibrils and has high mechanical strength such as tensile strength, stretch resistance and elasticity. This mechanical strength is increased by the entanglement of the microfibrils, and the orientation is imparted to further increase the strength in that direction. Bacterial cellulose has the property of being easily oriented by pressing.

[0053]

【Example】

Example 1 Sucrose 5 g / dl, yeast extract (Difco) 0.
5 g / dl, ammonium sulfate 0.5 g / dl, KH ₂ PO ₄ 0.3 g
_{/ Dl, MgSO 4 · 7H 2} O 0.05g / dl (pH
50 ml of the medium having the composition of 5.0) was placed in a 200 ml Erlenmeyer flask, and steam sterilized at 120 ° C. for 20 minutes. In addition, 0.5 g / dl of yeast extract, 0.3 g / dl of peptone,
Acetobacter, aceti, subspice, xylinum ATC grown on a test tube slope agar medium having a composition of mannitol 2.5 g / dl (pH 6.0) (30 ° C., 3 days)
One platinum loop of C 10821 was inoculated and cultured at 30 ° C. After 30 days, a gel-like film containing white bacterial cellulosic polysaccharide was formed on the upper layer of the culture solution.

The gel film thus obtained was washed with water to about 1
The sample was spread to a thickness of cm and pressed with a test press (Tester Sangyo Co., Ltd.) at a pressure of about 10 kg / cm ² to squeeze out water. Stick this on a glass plate and
It was dried at 05 ° C for 2 hours to obtain a sheet having a thickness of about 10 µm.

The X-ray diffraction pattern of the obtained sheet is shown in FIG. This figure takes the axis of rotation parallel to the seat surface,
It is a diffraction image photographed by making X-rays incident at right angles to the rotation axis while rotating the sheet. As shown in the figure,
(B) 101 plane, are oriented either of (ii) 10 first surface and (iii) 002 plane, this sheet were those that extremely high levels of surface orientation.

The following table shows the results of measuring the elastic modulus of this sheet, the existing cellulosic sheet, and various polymer two-dimensional materials using a tensile tester.

[0057]

[Table 1] * 1 Polymetaphenylene isophthalamide sheet * 2 Biaxially oriented polyethylene terephthalate sheet

Example 2 The same gel-like bacterial cellulose as that used in Example 1 was unidirectionally rolled using a roll press (Yoshida Kogyo Co., Ltd.) and pressed. This product was also attached to a glass plate and dried at 105 ° C. for 2 hours to obtain a sheet.

The X-ray diffraction pattern of the obtained sheet is shown in FIG. This figure shows that the sheet is fixed and X is applied to the film surface.
It is a diffraction image photographed with a line incident vertically. In the figure, the arrow indicates the rolling direction. As shown in the figure, (a)
101 plane, (b) it found oriented in all 10 first surface and a 002 plane, uniaxial orientation is clear. Regarding the plane orientation, almost the same orientation as in Fig. 1 was recognized.

When the elastic modulus of this sheet was measured in the same manner as in Example 1, it was 20 GPa in the rolling direction.

Example 3 Bacterial cellulose was added to novoloid fibers (Kinol fiber KF0203, manufactured by Koriei Chemical Industry Co., Ltd., φ14 μm, 3 mm length), and a sheet having a basis weight of 60 g / m ² was made by the TAPPI method (TAPPI method). stand
ard T205m-58).

[0062] In addition, for comparison the normal wood pulp _(N.
U. SP) Highly those beaten (CSF245ml)
A mixed paper made of pulp and kainol fiber was also prepared.

The breaking lengths of these sheets were measured by a self-recording tensile tester. The results were as shown in the following table.

[0064]

[Table 2]

The use of bacterial cellulose made papermaking possible with a small amount of addition and increased strength. In the case of using ordinary pulp, it was impossible to make paper with 10 parts or less.

Example 4 Using various inorganic fibers, a mixed paper with BC was prepared and the breaking length was measured. The results are shown in the table below.

[0067]

[Table 3]

In each case, addition of 5 to 10% made papermaking possible.

Example 5 Gel-like bacterial cellulose was pressed and dried to obtain a sheet. The Young's modulus E measured by the vibration lead method was as follows. E = 13.6 GPa

This value is 5 to 10 times the normal Young's modulus of paper made of wood pulp alone.

Example 6 Gel-like bacterial cellulose was disaggregated by a homogenizer and paper was made by the TAPPI method. Young's modulus E measured by the vibration lead method was E = 7.4 GPa.

Further, for the purpose of improving drainage and improving the yield of fine particles, polyamide epichlorohydrin resin (Kaimen 557H manufactured by Dick Hercules) 5%
(Solid content ratio) was added and paper was made. The E of this paper was as follows: E = 8.1 GPa

In each case, high strength paper was obtained.

Example 7 Wood pulp (N.U.KP) (CSF540 ml) was added with bacterial cellulose (BC), and van sulphate ± 5% (solid content ratio) was added to prepare paper. The characteristics were as follows:

[0075]

[Table 4]

The paper strength was improved by the addition of BC.

Example 8 Gel-like bacterial cellulose obtained by predetermined culture was disintegrated by a standard pulp disintegrator and then filtered through a 125 mesh sieve to obtain a paste-like disintegrant having a solid content of about 8.8%. Obtained. This was used in the following experiments.

Photocrosslinkable Polyvinyl Alcohol (PVA)-
SbQ (GH-17SbQ 10.5wt% 1.2mo
1% Toyo Gosei Co., Ltd.), the above disaggregated material and water were mixed in a dark room at the ratio shown in the table below.

[0079]

[Table 5] (a) PVA-SbQ (GH17SbQ 10.5wt
% 1.2 mol%) (b) Bacterial cellulose disaggregated material (dry weight conversion) (c) H ₂ O

The mixture in the above table was cast on an acrylic plate with a glass rod to a thickness of about 0.7 mm. This was air-dried overnight and further air-dried at 40 ° C. for 30 minutes. The operation up to this point was performed in a dark room. This was exposed to sunlight for 30 minutes and crosslinked to obtain a sheet.

The above material was cut into a ribbon of 3 × 1 cm, put in water and subjected to a 2 hr swelling test. The results are shown in the table below.

[0082]

[Table 6]

Swelling could be prevented by adding bacterial cellulose.

Next, a tensile test of this sheet was conducted.

[0085]

[Table 7]

By adding bacterial cellulose, the elastic modulus could be improved by 1.3 to 1.6 times.

Example 9 A mixture having the composition shown in the table of Example 8 was sandwiched between two glass plates using a slide glass having a thickness of 1 mm as a spacer. This was directly exposed to sunlight for 30 minutes to be crosslinked to obtain a konjak gel in a wet state.
This was placed in distilled water and a swelling test was conducted in the same manner as in Example 8. The results are shown in the table below.

[0088]

[Table 8] * Samples 3 and 4 were destroyed by swelling.

Incorporation of bacterial cellulose prevented swelling and breakage of the gel.

Example 10 To 3.5 parts of dried bacterial cellulose was added 100 parts of dimethylacetamide, and the mixture was stirred under reflux for 60 minutes. Next, after cooling to 100 ° C. and gradually adding 10 parts of lithium chloride, the mixture was stirred at room temperature for one day to dissolve the bacterial cellulose. This spinning solution was spun with a coagulating solution of tetrahydrofuran and a spinning draft 1.5 bath length of 80 cm, and the yarn discharged from the spinning bath was stretched by 50% in water at 50 ° C. and then dried. The properties of the obtained fiber are as shown in the table below.
It was also spun Similarly for wood pulp _{(L. B.} KP) for comparison.

[0091]

[Table 9]

[0092] The bacterial cellulose was macerated in a mixture of Example 11 copper powder (Fukuda Metal Foil & Powder Co., Ltd. Fai10myuemu) and wood pulp _{(N. U. KP) (} CSF540ml) was added and paper making by TAPPI method. A mixed paper of copper powder and wood pulp was also prepared for comparison. The physical properties of these sheets were measured by an automatic recording tensile tester. The results are shown in the table below.

[0093]

[Table 10]

By using bacterial cellulose, there was no leakage of copper powder and the strength was greatly improved. In the case of ordinary pulp, 60% or more of the copper powder leaked out.

Example 12 Disaggregated bacterial cellulose (BC) was added to wood pulp (NUKP) (CSF540 ml), and TA was added.
Paper was made by the PPI method. The paper was impregnated with a phenol resin, air-dried, and hot pressed to produce a phenol laminate.

For comparison, a phenol laminate made of wood pulp alone was similarly prepared. These phenol laminated plates were molded into dumbbell No. 1 type (JIS K-7113), and physical properties were measured by an automatic recording type tensile tester.
The results are shown in the table below.

[0097]

[Table 11]

The use of bacterial cellulose greatly improved the strength of the phenol laminate.

Example 13 The same gel-like bacterial cellulose used in Example 1 was hot-pressed at 150 ° C. and 5 kg / cm ² for 5 minutes (Yoshida Kogyo Co., Ltd.) to obtain a sheet. The obtained sheet is provided with polyethyleneimine-treated polyethylene film 320
A laminate film was produced by laminating at ℃. The physical properties of this laminated film were measured by an automatic recording type tensile tester. As a result, a laminated film having an elastic modulus of 16.2 GPa, which was significantly higher than the elastic modulus of 1.7 GPa of the ordinary cellophane-polyethylene laminated film, was obtained.

Example 14 Silicon nitride and silicon carbide (manufactured by Tateho Chemical Co., Ltd., 10 μm long)
Bacterial cellulose (BC) that had been disaggregated was added to and paper was made by the TAPPI method. Further, for comparison, a mixed paper with wood pulp (NUKP) and microfibril cellulose (MFC: manufactured by Daicel Chemical) was also similarly prepared. The physical properties of the obtained sheet were measured by an automatic trial type tensile tester. The results are shown in the table below.

[0101]

[Table 12]

By using bacterial cellulose, there was no leakage of silicon nitride and silicon carbide and the elastic modulus was greatly improved. In the case of ordinary pulp, 60% or more of silicon nitride and silicon carbide leaked out, and in the case of MFC, most of them leaked out.

Example 15 Fumaric acid 2 g / dl, potassium dihydrogen phosphate 0.2 g /
dl, manganese sulfate tetrahydrate 1 mg / dl, magnesium sulfate heptahydrate 1 mg / dl, calcium chloride 0.05 g /
A liquid medium having a composition of dl, yeast extract (Difco) 1.0 g / dl and peptone (Difco) 1.0 g / dl was adjusted to pH 7.0 with ammonia. 50 this medium
Dispense 50 ml each into a 0 cc Sakaguchi flask and
li ATCC 11775 inoculated with one platinum loop each 3
The cells were cultured at 0 ° C for 24 hours. The cells were collected by centrifugation according to a conventional method. After washing twice with physiological saline,
The suspension was suspended in the same amount of physiological saline as the wet weight.

An attempt was made to immobilize the cells on a carrier made of gelatin + the present cellulose by the following method. As the immobilization method, the immobilization method using transglutaminase described in JP-A-59-66886 was used. Gelatin (Miyagi Kagaku) and the disaggregated product of the bacterial cellulose were mixed in the composition shown in the table below. The cells were further mixed in this mixed solution so that the concentration was 3.5%, and then 0.1 unit of transglutaminase was added to 1 mg of gelatin according to the method described in JP-A-59-66886. The mixture was allowed to stand at 25 ° C. for 1 hour for gelation. This gel was cut into a 5 mm square die and added to the reaction solution to examine the activity of aspartase. The reaction solution, fumaric acid _{20g / dl, 1mM · MgSO 4} · 7
The solution containing H ₂ O was adjusted to pH 8.5 with ammonia.
It adjusted and used. The bacterial cell concentration was 0.
A gelatin gel with immobilized microbial cells was added to the reaction solution so as to be 5%. The reaction was carried out for 1 hour, and the aspartic acid concentration was quantified by colorimetry with ninhydrin every 5 minutes to determine the initial reaction rate. The number of bacteria leaked into the reaction solution was determined by colony counting 30 minutes after the reaction was started. The results are shown in the table below.

[0105]

[Table 13]

The enzyme activity was determined from the initial reaction rate. The enzyme activity was expressed as a relative value, with 100 being the first time when bacterial cellulose was not added. The reaction was repeated 10 times. The results are shown below.

[0107]

[Table 14]

Since the bacterial cells were prevented from leaking out by adding the bacterial cellulose, it became possible to maintain the activity after repeated use.

Further, the breaking strength of the same gelatin as that shown in the above table and the gelatin to which the reinforcing material was added was measured. The measurement was performed by gelling the gelatin solution in a cylindrical plastic container having a diameter of 22 cm, and then setting it in a rheometer (Fudo Kogyo Co., Ltd., NRM2002J).
The maximum load when the mmφ adapter was directly penetrated into the gel was expressed as the breaking strength. The results are shown in the table below.

[0110]

[Table 15]

Bacterial cellulose was recognized to have a reinforcing effect superior to that of conventional reinforcing materials.

Example 16 For immobilization on alginate gel, sodium alginate and bacterial cellulose were mixed according to the table below. Then, according to a conventional method, the mixture was added dropwise to a 0.1 M CaCl ₂ solution to obtain a jade-shaped gel.

As for the reaction conditions and the like, the reaction of aspartase was carried out as in Example 15. The number of leaked bacteria was determined by the number of colonies after 30 minutes without changing the reaction solution. The results are shown in the table below.

[0114]

[Table 16]

The above reaction solution was replaced every 15 minutes, and a total of 4 times of repeating reaction of the immobilized carrier. The enzyme activity was determined from the initial rate by measuring the aspartic acid concentration every 3 minutes. The results are shown in the table below. Aspartase activity is 1 at the first time when the cellulosic material is not added.
It was shown as a relative value when it was set to 00.

[0116]

[Table 17]

As a result of adding bacterial cellulose as a reinforcing material, the leakage of bacterial cells was prevented, so that the high-strength immobilization carrier of the present invention can be repeatedly used rather than the conventional immobilization carrier.

Example 17 Invertase was immobilized on a photocrosslinkable resin by the method described below. 1 part of invertase and phosphate buffer (pH
6.0) 2 parts mixed with a photo-crosslinkable resin solution (pH
60) 20 parts were mixed. This mixed solution was cast on a glass plate, air-dried for 1 day, and then irradiated with light for 1 hour to crosslink and cure. The reinforcing material was added so that the final concentration was 5%.

This immobilization membrane was cut into a size of 5 × 5 mm, and stirred in 40 ml of a solution of 4 g of sucrose at 40 ° C. for 24 hours, and the decomposition rate of sucrose was examined. The reaction was repeated 10 times. The breaking stress and elastic modulus were also investigated.
The results are shown in the table below.

[0120]

[Table 18] The addition of bacterial cellulose greatly improved the physical properties of the immobilized enzyme membrane, such as membrane rupture stress and elastic modulus. Therefore, the operation for immobilizing the enzyme was facilitated, and the time required for the operation was shortened to 3/4 when the cellulosic material was added, as compared with the case where the cellulosic material was not added.

[0121]

The high mechanical strength molding material of the present invention is excellent in tensile strength, stretch resistance, elasticity and the like. In particular, the sheet obtained by pressing and drying has a very high elastic modulus, and in the example product, the elastic modulus of the sheet of polymetaphenylene isophthalamide having the highest elastic modulus among the currently known two-dimensional materials. It was more than double.

Therefore, this material is used as a reinforcing material for composite plastics required to have high strength, for example, as a body material for ships, aircraft, automobiles, as a wiring board, or for high-quality paper such as recording paper and vibration of percussion instruments. It can be used as a board.

In addition, this material is a natural product, does not cause inflammation on human skin, and is excellent in breathability, so that it is an excellent base material for Van Ricoh.

Further, it is excellent against breakage and deformation due to physical stress and is also strong against breakage and deformation due to swelling caused by a solution or the like. Therefore, when it is used as an immobilization carrier, not only can it be vigorously stirred and packed into a very long column, but also a carrier that is conventionally too weak and unusable can be newly added as an immobilization carrier. It can be used. When this immobilizing carrier is produced, there is no aggregation of the active ingredient that frequently occurs in the conventional immobilizing carrier producing process, and therefore, the active ingredient can be uniformly dispersed in the immobilizing carrier.

[Brief description of drawings]

FIG. 1 is an electron micrograph of a cellulosic material produced by Acetobacter acetate subsp. Xylinum.

FIG. 2 shows an X-ray diffraction pattern of the product of the present invention.

FIG. 3 shows an X-ray diffraction pattern of the product of the present invention.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] May 19, 1995

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction content]

[Document name] Statement

Title of invention Immobilized carrier reinforcement

[Claims]

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immobilizing carrier reinforcing material made of a specific cellulose produced by bacteria.

[0002] By adding this reinforcing material to the solid support, the bacterial cells, enzymes and the like can improve the strength of the immobilizate and reduce the leakage of bacterial cells during use.

[0003]

2. Description of the Related Art Conventionally, agar, carrageenan alginic acid, gelatin, collagen, polyamino acid, photocrosslinkable resin, polyacrylamide and various resins have been used as an immobilization carrier. Polyvinylidene chloride, polyester, etc. By mixing these carriers as a reinforcing material, it has been attempted to improve the strength and spin adhesive.

As the cellulose produced by bacteria, Acetobacter xylinum (Acetoba) is used.
It is known to use a sheet-shaped product produced by Cter xylinum) ATCC 23769 for a medical pad (Japanese Patent Laid-Open No. 59-120159).

[0005]

However, in this immobilization product, the reinforcing material enlarges the carrier to reduce the surface area per unit area, which inhibits substrate diffusion in the carrier or is immobilized on the carrier. There has been a problem that the reaction rate and the reaction yield are lowered due to the relative decrease in the concentrations of the enzyme, the substance of biological origin, the catalyst, and other reactive substances (hereinafter referred to as “active ingredients”). In addition, if the active ingredient is used for a long period of time or repeatedly because it leaks from the immobilized carrier,
There is a problem that the activity gradually decreases, and it is difficult to prevent the leakage of the active ingredient from the carrier even if a conventional reinforcing material such as polyvinylidene chloride or polyester is added. Of the conventional immobilization carriers, a very weak gel, for example, a 6% aqueous solution of photo-crosslinkable polyvinyl alcohol cross-linked, when used as an immobilization carrier, it is fixed because water causes swelling. There is also a problem that the carrier is destroyed.

Although the above-mentioned examples utilize cellulose produced by bacteria, its use is limited to medical pads, and it is completely known that it has high utility value as a material in the field of high mechanical strength. Didn't.

The object of the present invention is to improve strength, prevent sticking,
Another object of the present invention is to provide a high-strength molding material that can be used as an immobilization carrier for enzymes, microorganisms, etc., that has a function of preventing the leakage of cells and the like.

[0008]

Means for Solving the Problems The present inventors have conducted various studies in order to achieve these objects, and a cellulose composed of ribbon-shaped microfibrils produced by a microorganism has extremely large mechanical strength such as tensile strength, Further, they have found that the above-mentioned object can be achieved by using this bacterial cellulose as an immobilizing carrier reinforcing material, which is also excellent in the retention of bacterial cells, and the present invention has been completed based on this finding.

That is, the present invention relates to an immobilizing carrier reinforcing material composed of bacterial cellulose composed of ribbon-shaped microfibrils.

As shown in the electron micrograph of FIG. 1, bacterial cellulose is composed of ribbon-shaped microfibrils having a width of 100 to 500Å and a thickness of 10 to 200Å. Generally, it is obtained in the form of gel and has a water content of 9
It is 5% (w / v) or more.

This cellulose is easily decomposed by cellulase to produce glucose. That is, a cellulase (EC) was added to a 0.1% (w / v) suspension of the present cellulose.
3,2,1,4) (manufactured by Amano Pharmaceutical Co., Ltd.) was dissolved to 0.5% (w / v), and the solution was dissolved in 0.1 M acetate buffer at 30 ° C. for 24 hours.
Allowed to react for hours. As a result, it was observed that part of this substance was decomposed, and when the supernatant was developed by paper chromatography, small amounts of cellobiose, cellotriose and other cellooligosaccharides were detected in addition to glucose. In addition to this, small amounts of fructose, mannose, etc. were sometimes detected.

That is, the bacterial cellulose of the present invention includes cellulose and a heteropolysaccharide having cellulose as a main chain and glucans such as β-1,3 and β-1,2. In the case of the heteropolysaccharide, the constituent components other than cellulose are hexoses such as mannose, fructose, galactose, xylose, arabinose, rhamnose and glucuronic acid, pentose sugars and organic acids. These polysaccharides may be a single substance, or two or more polysaccharides may be mixed due to hydrogen bonding or the like.

Any bacterial cellulose can be used as long as it is as described above.

Microorganisms producing such bacterial cellulose are not particularly limited, but Acetobacter aceti subsp. Xylinum (Acetobacter)
eraceti subsp xylinum) ATCC
10821 or the same Pastourian (A. pas
teurianus), the same license (A. rance)
ns), Sarcina ve
ntriculi), Bacterium xyloides (B
It is possible to use bacteria that produce bacterial cellulose, such as bacteria of the genus Pseudomonas, Pseudomonas, and bacteria of the genus Agrobacterium.

As a method for culturing these microorganisms to produce and accumulate bacterial cellulose, a general method for culturing bacteria may be used. That is, a carbon source, a nitrogen source, an inorganic salt, and other amino acids as necessary, an ordinary nutrient medium containing organic micronutrients such as vitamins is inoculated with a microorganism,
Stir or gently ventilate. As the carbon source, glucose, sucrose, maltose, starch hydrolyzate, molasses, and the like are used, and ethanol, acetic acid, citric acid, and the like can be used alone or in combination with the above-mentioned sugars. As the nitrogen source, ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, nitrates, organic or inorganic nitrogen sources such as urea and peptone are used. As the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like are used. As organic micronutrients, amino acids, vitamins, fatty acids, nucleic acids, and peptones, casamino acids, yeast extracts, and soybean protein hydrolysates containing these nutrients are used, and auxotrophic mutations that require amino acids for growth are used. When using strains, it is necessary to further supplement the required nutrients.

The culture conditions may be normal, and the pH is 5 to 9
Then, while controlling the temperature to 20 to 40 ° C, 1 to 3
If cultivated for 0 days, bacterial cellulose accumulates on the surface in the form of a gel.

The bacterial cellulose used in the present invention may be a purified product isolated from a culture of a microorganism, or may contain impurities to some extent depending on the use. For example, residual sugar, salts, yeast extract, etc. in the culture solution may remain in the microbial cellulose. In addition, the cells may be contained to some extent.

The gel is taken out and washed with water if necessary. A chemical such as a bactericide or a pretreatment agent can be added to the washing water according to the purpose.

In place of the conventional immobilizing carrier reinforcing material, this bacterial cellulose is left as it is or is disintegrated,
Alternatively, by exposing and drying these substances, it is possible to obtain an unprecedented high-strength immobilized carrier.

The following method is available for producing such an immobilization carrier. Basically, the bacterial cellulose and the following immobilized carrier material are mixed and then gelled,
It may be polymerized or molded. If necessary, the active ingredient for the purpose of immobilization may be added together for immobilization during the mixing, or the active ingredient may be immobilized after the immobilization carrier is manufactured.

The carrier material is not particularly limited as long as it can be mixed with the bacterial cellulose, but the following materials can be used, for example. Agarose, dextran, cellulose, cellulose derivatives, alginic acid,
Alginate, chitin, chitosan, collagen, albumin, amino acid polymer, polystyrene, polyacrylamide, tannin, silicone rubber, casein, agar, carrageenan, polyurethane, poly-2-hydroxyethylmethacrylic acid, polyvinyl chloride, γ-methylpolyglutamic acid, Polyvinylpyrrolidone, polydimethylacrylamide, photo-crosslinkable resin, polyethylene glycol derivative, polypropylene glycol derivative, polybutadiene derivative, collodion, nylon, polyurea, silica gel, silicon derivative, phenylsiloxane, fibrin, cellulose nitrate, hydrocarbon, phospholipid, calcium phosphate gel , Phenoxyacetylated products, glucomannan, etc.

Further, photocrosslinkable polyvinyl alcohol is said to have a better affinity for living organisms than conventional photocrosslinkable resins, and it is thought that the use of immobilizing agents for enzymes, microorganisms, etc. will be further improved by this. ing. However, in this case, there is a problem that the photocrosslinkable polyvinyl alcohol is swollen by water and the crosslinked structure is destroyed.
However, this swelling can be prevented by adding bacterial cellulose.

The method for producing the immobilization carrier will be described below.
When the bacterial cellulose is in a gel form as it is produced by microorganisms, it is a dried product obtained by drying the gel form, or a dry product obtained by disintegrating and drying the gel form. If the material does not maintain the fibrous form, the carrier material is made into a solution or melted with an appropriate solvent so as to have fluidity, and then impregnated into the bacterial cellulose. An immobilization carrier is obtained by gelling. The method of gelation varies depending on the carrier material. For example, in the case of sodium alginate, it may be mixed and then added in a calcium chloride solution, and in the case of agar, the temperature may be lowered.

When the bacterial cellulose is in a disaggregated state, or in a dried state in which a fibrous form obtained by a method such as freeze-drying or critical point drying remains, the bacterial cellulose and the carrier material are used. Differently from the above-mentioned impregnation, the immobilized carrier is obtained by gelling, polymerizing or molding after mixing by an ordinary method.

The concentration of the bacterial cellulose in the immobilization carrier is 0.01% to 99%, preferably about 0.1 to 2%.

The shape of the immobilization carrier obtained by the above method can be freely selected according to the type and method of the reaction. For example, when it is packed in a column or put in a stirring tank, it may be processed into a ball shape or may be formed into a rod shape or a film shape if necessary. When the immobilized carrier is produced by mixing the disaggregated bacterial cellulose with a carrier material and then gelling, polymerizing or molding the immobilized carrier, the immobilized carrier may be processed and molded in the same manner as in the conventional method.

On the other hand, in the case of directly producing the immobilized carrier from the gel form as produced by the microorganism, the carrier material may be impregnated with the gel-like bacterial cellulose and then gelled. In order to process it into a predetermined shape, the gel-like bacterial cellulose may be processed into a required shape and then impregnated with a carrier material, while the immobilized carrier may be manufactured and then processed into a required shape. good.

The active ingredient immobilized on the immobilization carrier of the present invention is an enzyme, a microorganism, a substance derived from a living body, a catalyst or other reactive substance, and any substance generally used may be used. Examples of active ingredient enzymes include aspartase, L-aspartate β-decarboxylase, L-leucine dehydrogenase, hydantoinase, DL-2-amino-Δ.
² -thiazoline-4-carboxylic acid hydrolase, dihydropyrimidinase, acylase acting on acylamino acid,
Tryptophanase, tryptophan synthetase, tyrosinase, fumarase, histidine ammonia lyase, L-amino acid oxidase, α-amylase, β-
Amylase, invertase, glucose isomerase, penicillin acylase, cephalosporin acylase, 5'-AMP deaminase, arginine deiminase, aldolase, cysteine desulfhydrase, methioninase, phosphodiesterase, chymotrypsin, trypsin, papain, naringinase, lactase, glucoamylase, Leucine aminopeptidase,
Pepsin, aminolactam hydrolase, amylolactam racemase, glutamate dehydrogenase, aspartate decarboxylase, pullulanase, hyaluronidase, 1,4-α-glucan phosphorylase, cyclodextrin glycosyltransferase, dextran sucrase, α-glucosidase, β-glucosidase, hexokinase, Δ ¹ -dehydrogenase, 11β
-Hydroxylase, 20β-dehydrogenase, 3β-dehydrogenase, Δ ¹ -hydroxylase, steroid esterase,
5'phosphodiesterase, ATP deaminase, acetate kinase, adenylate kinase, adenosine kinase,
Carbamyl phosphokinase, pyruvate kinase, glycolytic enzyme chymosin, alkaline protease, rennin, pronase, protease, catalase, lysozyme, D
-Oxynitrase, alcohol dehydrogenase, glucose-6-phosphate dehydrogenase, nitrate reductase, nitrite reductase, rhodanese, glutaminase, uricase, peroxidase, lipase, cholesterol oxidase, penicillinase, alkaline phosphatase, acid phosphatase, acetylcholinesterase, and the like. To be Examples of microorganisms of the active ingredient include Brevibacterium ammoniagenes, Escherichia coli, Erviherbicola, Streptococcus faecalis, Pseudomonas putida, Serratia.
Macerans, Bacillus subtilis, Acetobacter
Aceti, Lactobacillus delbrettii, Pseudomonas fluorescens, Micrococcus luteus,
Bacillus megaterium, Penicillium chlorigenum, Candida tropicalis, Saccharomyces cerevisiae, etc. are mentioned. In addition, animal cells, plant cells, etc. may be immobilized if necessary.

The thus obtained immobilization product can be used for the production of useful substances such as medicines and foods using microorganisms and enzymes. It can also be used as a bioreactor in analytical and industrial production processes.

The catalyst and other reactive substances may be organic or inorganic substances which can coexist with the bacterial cellulose and are used in ordinary chemical reactions. These materials can be packed in a column and used as a reaction bed, or can be used for various chromatography. On the other hand, the immobilized carrier can be used for physically separating and selecting or purifying the target substance by selecting the particle size and size.

[0031]

Example 1 Sucrose 5 g / dl, yeast extract (Difco) 0.
5 g / dl, ammonium sulfate 0.5 g / dl, KH ₂ PO ₄ 0.3 g
_{/ Dl, MgSO 4 · 7H 2} O 0.05g / dl (pH
50 ml of the medium having the composition of 5.0) was placed in a 200 ml Erlenmeyer flask, and steam sterilized at 120 ° C. for 20 minutes. In addition, 0.5 g / dl of yeast extract, 0.3 g / dl of peptone,
Acetobacter, aceti, subspice, xylinum ATC grown on a test tube slope agar medium having a composition of mannitol 2.5 g / dl (pH 6.0) (30 ° C., 3 days)
One platinum loop of C 10821 was inoculated and cultured at 30 ° C. After 30 days, a gel-like film containing white bacterial cellulosic polysaccharide was formed on the upper layer of the culture solution.

The gel-like bacterial cellulose thus obtained was disintegrated with a standard pulp disintegrator and then 125 mesh.
The mixture was filtered through a No. 1 sieve to obtain a paste-like disaggregated product having a solid content of about 8.8%. This was used in the following experiments.

Fumaric acid 2 g / dl, potassium dihydrogen phosphate 0.2 g / dl, manganese sulfate tetrahydrate 1 mg / dl,
Magnesium sulfate heptahydrate 1 mg / dl, calcium chloride 0.05 g / dl, yeast extract (Difco) 1.0 g /
A liquid medium having a composition of dl and peptone (Difco) 1.0 g / dl was adjusted to pH 7.0 with ammonia. 50 ml each of this medium was dispensed into a 500 cc Sakaguchi flask, one platinum loop of E. coli ATCC 11775 was inoculated, and the mixture was cultured at 30 ° C. for 24 hours. The cells were collected by centrifugation according to a conventional method. 2 with saline
After washing twice, it was suspended in the same amount of physiological saline as the wet weight.

An attempt was made to immobilize the cells on a carrier made of gelatin + the present cellulose by the following method. As the immobilization method, the immobilization method using transglutaminase described in JP-A-59-66886 was used. Gelatin (Miyagi Kagaku) and the disaggregated product of the bacterial cellulose were mixed in the composition shown in the table below. The cells were further mixed in this mixed solution so that the concentration was 3.5%, and then 0.1 unit of transglutaminase was added to 1 mg of gelatin according to the method described in JP-A-59-66886. The mixture was allowed to stand at 25 ° C. for 1 hour for gelation. This gel was cut into a 5 mm square die and added to the reaction solution to examine the activity of aspartase. The reaction solution, fumaric acid _{20g / dl, 1mM · MgSO 4} · 7
The solution containing H ₂ O was adjusted to pH 8.5 with ammonia.
It adjusted and used. The bacterial cell concentration was 0.
A gelatin gel with immobilized microbial cells was added to the reaction solution so as to be 5%. The reaction was carried out for 1 hour, and the aspartic acid concentration was quantified by colorimetry with ninhydrin every 5 minutes to determine the initial reaction rate. The number of bacteria leaked into the reaction solution was determined by colony counting 30 minutes after the reaction was started. The results are shown in the table below.

[0035]

[Table 1]

The enzyme activity was determined from the initial reaction rate. The enzyme activity was expressed as a relative value, with 100 being the first time when bacterial cellulose was not added. The reaction was repeated 10 times. The results are shown below.

[0037]

[Table 2]

Since bacterial cells were prevented from leaking out by adding bacterial cellulose, it was possible to maintain the activity after repeated use.

Further, the breaking strength of the same gelatin as that shown in the above table and the gelatin to which the reinforcing material was added was measured. The measurement was performed by gelling the gelatin solution in a cylindrical plastic container having a diameter of 22 cm, and then setting it in a rheometer (Fudo Kogyo Co., Ltd., NRM2002J).
The maximum load when the mmφ adapter was directly penetrated into the gel was expressed as the breaking strength. The results are shown in the table below.

[0040]

[Table 3]

Bacterial cellulose was found to have a reinforcing effect superior to that of conventional reinforcing materials.

Example 2 For immobilization on alginate gel, sodium alginate and bacterial cellulose were mixed according to the table below. Then, according to a conventional method, the mixture was added dropwise to a 0.1 M CaCl ₂ solution to obtain a jade-shaped gel.

The reaction conditions and the like were the same as in Example 15 to carry out the reaction of aspartase. The number of leaked bacteria was determined by the number of colonies after 30 minutes without changing the reaction solution. The results are shown in the table below.

[0044]

[Table 4]

The above reaction solution was replaced every 15 minutes, and a total of 4 times of repeating reaction of the immobilized carrier was carried out. The enzyme activity was determined from the initial rate by measuring the aspartic acid concentration every 3 minutes. The results are shown in the table below. Aspartase activity is 1 at the first time when the cellulosic material is not added.
It was shown as a relative value when it was set to 00.

[0046]

[Table 5]

As a result of the addition of bacterial cellulose as a reinforcing material, the leakage of bacterial cells was prevented, so that the high-strength immobilization carrier of the present invention can be repeatedly used rather than the conventional immobilization carrier.

Example 3 Invertase was immobilized on a photocrosslinkable resin by the method described below. 1 part of invertase and phosphate buffer (pH
6.0) 2 parts mixed with polyvinyl alcohol (PVA) -SbQ (GH-17 Sb) as a photo-crosslinkable resin.
Q10.5wt% 1.2mol%, Toyo Gosei Co., Ltd.)
20 parts (pH 6.0) were mixed. This mixed solution was cast on a glass plate, air-dried for 1 day, and then irradiated with light for 1 hour to crosslink and cure. The reinforcing material was added so that the final concentration was 5%.

The immobilization membrane was cut into a size of 5 × 5 mm, and stirred in 40 ml of a solution containing 4 g of sucrose at 40 ° C. for 24 hours while stirring to examine the decomposition rate of sucrose. The reaction was repeated 10 times. The breaking stress and elastic modulus were also investigated.
The results are shown in the table below.

[0050]

[Table 6]

The addition of bacterial cellulose greatly improved the physical properties of the immobilized enzyme membrane, such as membrane rupture stress and elastic modulus. Therefore, the operation for immobilizing the enzyme becomes easy, and the time required for the operation is 3 /
Shortened to 4.

[0052]

INDUSTRIAL APPLICABILITY The reinforcing material of the present invention is excellent against breakage and deformation due to physical stress and is also strong against breakage and deformation due to swelling caused by a solution or the like. Therefore,
Not only is it possible to perform vigorous agitation and packing into a very long column when compounded as a reinforcing material in an immobilized carrier,
A carrier that is conventionally too weak to be used can be newly used as an immobilized carrier. When this immobilizing carrier is produced, there is no aggregation of the active ingredient that frequently occurs in the conventional immobilizing carrier producing process, and therefore, the active ingredient can be uniformly dispersed in the immobilizing carrier.

[Brief description of drawings]

FIG. 1 is an electron micrograph of a cellulosic material produced by Acetobacter acetate subsp. Xylinum.

[Procedure Amendment 2]

[Document name to be corrected] Drawing

[Correction target item name] All drawings

[Correction method] Change

[Correction content]

FIG.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication H01B 3/48 (72) Inventor Shigenobu Mitsuhashi 2-709 Azuma Sakuramura, Shinji-gun, Ibaraki (72) Invention Kunihiro Ichimura 5-630 Matsushiro, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture (72) Inventor Shigeru Yamanaka 3-40-13 Ooka, Minami-ku, Yokohama-shi Kanagawa (72) Inventor Watanabe Otohiko Kawasaki-ku, Kawasaki-shi, Kanagawa Kannon 2-20-8 (72) Inventor Mio Nishi 6-735 Kita-Shinagawa, Shinagawa-ku, Tokyo Sony Corporation (72) Inventor Uryu Masaru 6-35 Kita-Shinagawa, Shinagawa-ku, Tokyo Soni -Inside the corporation

Claims (1)

[Claims] 1. An immobilization carrier reinforcement comprising bacterial cellulose comprising ribbon-shaped microfibrils.