JPH056560B2 - - Google Patents

Description

[Detailed description of the invention]

(a) Industrial application field This invention relates to an immobilized product and a method for producing the same. More details include enzymes, antigens, antibodies, microorganisms,
It is made by immobilizing a substance containing a peptide agent such as bacteria,
This article relates to immobilized materials useful as bioreactors for various analyzes and bioreactors for synthesis. (b) Prior Art Recently, immobilized enzymes, which are immobilized substances containing peptide agents such as enzymes on glass carriers, have come to be used as bioreactors for diagnosis and synthesis. As a method for producing these immobilized products, the surface of SiO ₂ glass previously obtained by a melting method is treated with alkali to generate hydroxyl groups, for example, an aminoalkyl group is introduced into this, and an enzyme is added to this. A method of fixing it is known and has been put to practical use. However, the conventional method described above requires a step to generate hydroxyl groups on the glass surface, and the amount of hydroxyl groups that can be generated per unit area is limited. It was difficult to obtain highly immobilized products. On the other hand, immobilized products using synthetic polymers such as nylon, polystyrene, polyacrylamide, etc. as carriers instead of glass are also known, but these have weak heat resistance and chemical resistance, and have problems with mechanical strength. It was hot. Regarding these points, the inventors of the present invention first discovered that if a gel obtained by hydrolyzing metal alkoxide is used as a support for immobilization, high activity and durability can be achieved without the step of introducing hydroxyl groups. We have found that a good immobilized product can be obtained. This invention was made by further developing the above knowledge. (c) Purpose The purpose of the present invention is to provide an immobilized product that can be produced in a fecal form and has a higher activity than conventional ones. (d) Configuration Thus, according to the present invention, the peptide-containing substance is a metal hydroxide compound and/or
or a condensate thereof, provided is an immobilized product that is immobilized on a gel body. The organometallic chelate compound in this invention includes various oxygen-containing organometallic chelate compounds used in the fields of chelate titration and organic synthesis, such as metal chelate compounds of alkadione or its derivatives. More specific examples include 2,4-bentanedione (acetylacetone), 3-phenyl-2,4-bentanedione (3-phenylacetylacetone), 2,4-hexanedione, 2,4 (or 3, 5)-heptanedione, 2,2,6,6-tetramethyl-3,5-heptanedione) dipipparoylmethane), and other metal chelate compounds of lower alkali diones, and the metals to be chelated include: Examples include calcium, aluminum, titanium, zinc, iron, zirconium, potassium, and the like. Among these, it is usually preferable to use aluminum acetylacetone and titanium acetylacetone. In obtaining the immobilized product of the present invention, the organometallic chelate compound is first converted into a gel by hydrolysis. This hydrolysis is carried out by bringing the organometallic chelate compound into contact with water, and is usually suitably carried out by mixing an organic solvent solution of the chelate compound with water. As this organic solvent, it is suitable to use a hydrophilic and solvent material, such as lower alcohols such as methanol and ethanol. Upon contact with water, organometallic chelate compounds begin to gradually hydrolyze. While the hydrolysis is progressing, it is suitable to maintain the mixture at a temperature of about 5 to 45°C to dissipate by-product lower alcohols in order to efficiently obtain the desired gel body. However, if the by-product alcohol evaporates naturally at room temperature, there is no particular need to heat it. As the hydrolysis progresses, a corresponding metal hydroxide compound is produced, and a gel body consisting of the metal hydroxide compound and/or its low condensate is usually obtained in about 1 minute to 12 hours. This gel body may be formed into dispersed particles under stirring. This gel body is glass-like and unstable, and usually requires sufficient drying (50 to 200
After isolation, the product is ground to an appropriate size and used as a carrier for the immobilized product. Depending on the case, it may be used as is or after being molded into an appropriate shape. However, such a gel body can be produced by coating an organometallic chelate compound undergoing hydrolysis on a suitable support (e.g., glass tube, metal tube, glass particles, etc.) so that it forms a film thereon. can. A gel body in the form of a membrane containing such a support is also suitably used as a carrier for immobilization, and forming a membrane of the gel body means such an embodiment. For example, as an organometallic chelate compound, aluminum acetylacetone [Al
(CH ₃ COCH ₂ COCH ₃ ) ₃ ], a glass-like gel body is obtained, but its composition is composed of Al(OH) ₃ or its low condensate [Al(OH) ₃ gel] , it has a much larger number of hydroxyl groups than the hydroxyl groups introduced into conventional SiO ₂ glass by alkali treatment. Therefore, it is possible to immobilize a larger amount of oxygen, etc. compared to conventional carriers. Of course, the same applies to other similar organometallic chelate compounds. During the above hydrolysis, the rate of hydrolysis can be accelerated by adding a small amount of mineral acid, but in particular, adding a small amount of hydrofluoric acid to the system can increase the activity of this hydroxyl group and increase the gelling rate. It was also discovered that the porosity of the body itself was further improved and a larger amount of oxygen, etc. could be immobilized. The amount of hydrofluoric acid added at this time is suitably 0.1 to 1.0 mol when expressed as a molar ratio to 1 mol of the organometallic chelate compound. The effect of improving the activity of the hydroxyl group is as follows:
This is thought to be mainly due to the effect of fluorine atoms directly bonded to metal elements in the gel. The gel body thus obtained is glass-like and microscopically porous, and is suitably used for immobilizing peptide-containing substances such as oxygen. The peptide-containing substance is usually immobilized on the gel body using a silane coupling agent. This silane coupling agent can be a silane derivative known in the art having a functional group such as an amino group, a thiol group, or an epoxy group, and specifically, γ-aminopropyltriethoxysilane, γ-aminopropyltriethoxysilane,
-talolopropyltrimethoxysilane, vinyltriethoxysilane, γ-glycidoxypropyltrimethoxysilane, N-β-(aminoethyl)-γ
-aminopropyltrimethoxysilane, etc. are used. Reactions with such silane coupling agents are carried out under conditions known in the art. For example, when using γ-aminopropyltriethoxysilane, dissolve this coupling agent in water to a concentration of about 10%.
After making an aqueous solution and adjusting the pH to 3 to 5, the sufficiently dried gel body is added to this solution or the formed gel body is kept in contact with the solution, mixed under heating, and treated for several hours. Obtained by washing with water to remove unreacted coupling agent. The above gel body into which the silane coupling agent is introduced has many coupling groups as a carrier compared to those introduced into conventional glass, and has a high reaction activity per oxygen and is a carrier for immobilized enzymes. It is useful as a column packing material. Peptide-containing substances such as enzymes, antigens, antibodies, microorganisms, and bacteria are immobilized on the thus treated gel body by a known method. For example, when using a gel body in which γ-aminopropyltriethoxysilane is used as a coupling agent and a large number of aminoalkyl groups are introduced into hydroxyl groups through ester bonds, a Schiff base in which aldehyde groups are formed using taraldehyde is introduced into the aminoalkyl groups. Immobilization can be carried out by contacting this with an enzyme, etc. to further form a Schiff base between the aldehyde group and the amino group of the enzyme, etc., and bonding.In addition, by diazotizing the aminoalkyl group, aromatic An amino group may be introduced and an enzyme etc. may be immobilized thereon, or a peptide bond may be directly formed between an aminoalkyl group and an enzyme etc. using cardioimide to immobilize it, depending on the type of enzyme etc. You can select it as appropriate. When using other coupling agents, enzymes and the like can be similarly immobilized directly or by appropriately converted coupling groups. However, immobilization can also be carried out without using a silane coupling agent by immobilizing an enzyme or the like on the OH group via the CN group using promo cyanide (BrCN). Examples of enzymes for immobilization include, but are not limited to, glucose oxidase, uricase, urease, creatininase, CoA-synthetase, CoA-oxidase, cholesterol oxidase, and cholesterol hydrolase. , antibodies, microorganisms, and bacteria can also be immobilized. The immobilized gel bodies (immobilized substances) obtained in this way are useful as bioreactors for diagnosis and synthesis in various forms, similar to immobilized substances using conventional glass carriers, and are also useful as bioreactors for diagnosis and synthesis. Compared to immobilized products, the amount of enzymes, etc. immobilized per carrier is large, and the capacity as a bioreactor is increased.
In addition, the organometallic chelate compound used also has a lower solvent solubility than metal alkoxides, which is advantageous in that it is easier to operate during production. (E) Example (Production of carrier - Step 1) Aluminum acetylacetone [Al
(CH ₃ COCH ₂ COCH ₃ ) ₃ ] 8g and 40ml of methanol
was added and stirred in a water bath at 80°C for 1 hour to uniformly dissolve. After adding and mixing 40 ml of water to this aluminum acetylacetonate solution, the solution was kept at 25°C in an open environment to proceed with hydrolysis, and the by-produced alcohol and methanol as a solvent were volatilized.
After some time, a white lumpy gel was obtained. This gel body was dried at about 500° C. for 5 minutes to obtain an Al(OH) ₃ gel body. (Aminoalkylation - Step 2) The gel obtained in Step 1 was crushed in a mortar and passed through a sieve to obtain beads of 100 to 200 meshes. A 5wt aqueous solution of γ-aminopropyltriethoxysilane was adjusted to pH 3.5 with 5N hydrochloric acid, and this solution
1 g of the above bead-shaped gel per ml was added, and the pH was further adjusted to 3.5. This mixture
The mixture was placed in a four-necked flask equipped with a stirrer, a thermometer, and a Dimroth, and the temperature was maintained at 75°C with a water bath, and the reaction was carried out for 3 hours with vigorous stirring. After the reaction was completed, the beads were transferred to a suction membrane filter, unreacted γ-aminopropyltriethoxysilane was removed with distilled water in step 1, and then dried in a desiccator to obtain an aminoalkylated gel. This aminoalkylated gel is stored in a desiccator. (Immobilization of oxygen - Step 3) The above aminoalkylated gel bodies (beads) were each treated with difunctional glutaraldehyde (25wt%).
The mixture was immersed in a phosphate buffer solution (pH 7.0) and reacted for about 30 minutes with stirring while reducing the pressure with an aspirator. Subsequently, the reaction was continued for about 30 minutes at normal pressure with stirring. The reaction temperature was 25°C. This was thoroughly washed with a phosphate buffer solution of pH 7.0 and dried. This treatment introduces Schiff base having an aldehyde group into the gel body. The obtained gel body was immersed in 1 mg/ml (glucose oxidase/phosphate buffer solution of pH 7.0), and the reaction was carried out by stirring gently under reduced pressure for 30 minutes at 25°C, and then for 60 minutes. The immobilization reaction was carried out under normal pressure with gentle stirring. Through this treatment, the amino groups of glucose oxidase participate in the reaction, and are further immobilized by forming a Schiff base with the aldehyde groups of the carrier (gel body). In this way, the glucose oxidase (1000 IU) immobilized product (beads) of the present invention was obtained. (Activity test) In order to compare the degree of immobilization of the above-mentioned immobilized product with that of the conventional method, an activity test was conducted using a voltammetric method. First, 10 ml of 0.1 wt% β-D(+) glucose solution (pH 7.0 phosphate buffer solution) was poured into a beaker containing 1 g of the immobilized beads of the present invention.
Stir gently for 15 minutes to react, and remove 9% from the reaction solution.
ml was taken and 1 ml of 5% potassium iodide solution was added. Through this operation, I ^- is oxidized to I ₂ (aq) with hydrogen peroxide generated by the enzymatic reaction. This I ₂
By reducing the enzyme by voltammetry and measuring the reduction wave, the amount of I ₂ can be quantified, and the degree of immobilization of the enzyme in the immobilized beads, that is, the degree of activity, can be determined. Table 1 shows the results of voltammetry using conventional immobilized beads using glass as a carrier and the immobilized beads of the present invention. In addition, the comparative example is Electro-
Aminopropyl CPG beads manufactured by Nucleonics were used.

[Table] Seconds.
In this way, the immobilized product of the present invention exhibits twice the activity of those using conventional glass as a carrier,
It is clear that the enzyme activity is high. (f) Effects As described above, the immobilized product of the present invention uses an L-form as a carrier that does not require a high-temperature heat treatment step, and does not require a step of introducing a hydroxyl group during immobilization, so it is superior to conventional products. It is advantageous in that it can significantly reduce costs and can be processed in an aqueous medium.
In addition, household chemicals with higher activity than conventional ones can be obtained, making them extremely useful industrially.

Claims (1)

[Scope of Claims] 1. A peptide-containing substance immobilized in a gel body consisting of a metal hydroxide compound and/or a condensate thereof produced by hydrolysis of an organometallic chelate compound having an oxygen atom as a ligand. Immobilized material. 2. One or more organometallic chelate compounds having an oxygen atom as a ligand are hydrolyzed to obtain a gel body, and a peptide agent-containing substance is added to the pulverized product or granules as a carrier using a coupling agent. A method for producing an immobilized product characterized by immobilization. 3 A solution of one or more organometallic chelate compounds having an oxygen atom as a ligand is applied to a support, and hydrolyzed there to form a gel film, and this gel film is used as a carrier. 1. A method for producing an immobilized product, which comprises immobilizing a peptide-containing compound using a coupling agent.