JPS6187700A - Peptide-containing substance immobilized material - Google Patents

Abstract

PURPOSE:The titled immobilized material useful for diagnosis of as a bioreactor for agriculture, producible simply at low cost, obtained by immobilizing a functional peptide-containing substance to zeolite as a carrier by chemical bond. CONSTITUTION:Zeolite is heated, sufficiently dried, ground, granulated, and treated with a silane coupling agent such as gamma-aminopropyltriethoxysilane, etc., to give zeolite treated with aminosilane. Then it is treated with glutaraldehyde, and a functional peptide-containing substance (e.g., beta-galactosidase, etc.) such as enzyme, antigen, antibody, etc., is immobilized to the zeolite by chemical bond, to give the aimed immobilized material.

Description

DETAILED DESCRIPTION OF THE INVENTION 0) Industrial Application Field This invention relates to a peptide-containing substance immobilized product. More specifically, the present invention relates to an immobilized peptide-containing substance useful as a diagnostic or industrial bioreactor in which a functional peptide-containing substance such as an enzyme, an antigen, or an antibody is immobilized on a carrier.

(B) Prior art Recently, various immobilized products have been proposed in which functional Begde-containing substances such as enzymes, antigens, and antibodies are immobilized on carriers.
In particular, so-called immobilized enzymes, in which biocatalysts such as enzymes are immobilized, are increasingly being used in diagnostic and industrial bioreactors.

There are various types of immobilization kits for such immobilized enzymes. Some examples include those in which the enzyme is retained, those in which the enzyme is closely held between membrane-like carriers, and those in which the enzyme is immobilized on the carrier by chemical bonding.

Among these, immobilized enzymes that are directly immobilized on a carrier by chemical bonding usually use glass particles or glass membranes as the carrier, and treat the surface with greed or strong alkali to introduce hydroxyl groups. In addition to this, it is possible to bind and immobilize enzymes using a silane coupling agent, etc. In addition, using a metal hydroxide-based porous gel body produced by hydrolysis of metal alkoxides, A method for similarly bonding and immobilizing an enzyme using this hydrophilic group is also known.

However, in such immobilized enzymes, it is necessary to prepare the carrier itself containing the aqueous group in advance by chemical treatment as described above, and since all companies use such chemical treatment, it is difficult to use bioreactors for food. When using it, there was a question of whether it was appropriate in terms of safety.

e) Purpose of the invention This invention was created in view of the above-mentioned problems of Jin's invention, and is easy to manufacture, low cost, and applicable to fields that require a high degree of safety, such as food bioreactors. The present invention also aims to provide a suitable peptide-containing substance immobilized product.

B) Structure of the Invention According to the present invention, there is provided an immobilized product in which a functional peptide-containing substance such as an enzyme, an antigen, or an antibody is immobilized on a carrier by chemical bonding, which is characterized in that Seviolite is used as the carrier. An immobilized substance containing substance is provided.

Seviolite used in this invention generally refers to a fibrous clay-like silica magnesium lump produced from natural minerals as a secondary mineral, and has a compositional formula of C0Ht)4cOH)a). dJlasi rtozo
・6~8H20 or Msr, Hl (Si 401
1) s・3H10 or S l +zMg 5oso
It is represented by (OH)4.

By using the above Seviolite as a carrier, the peptide-containing substance immobilized product of the present invention can be obtained. On this occasion,
After being shaped into a desired shape such as a granule or a film by Seviolite, it is used for immobilization of a peptide-containing substance. In this case, sepiolite can be physically shaped and used as a carrier for immobilization, but it is usually preferable to use one that has been sufficiently dried by heating. In particular, it is preferable to use a carrier from which adsorbed water and water of crystallization have been sufficiently removed by heating, since this improves the brittleness of the resulting carrier and thus the immobilized product, thereby making it possible to obtain a practical immobilized product. Such heat treatment is usually suitably carried out at a temperature of 450 to 550°C. At temperatures below 450°C, sufficient drying (dehydration) cannot be achieved even if heat treatment is performed for a long time;
If the temperature exceeds .degree. C., the amount of hydroxyl groups present in Seviolite will be reduced, so it is not suitable. Usually 450-55
It is most preferable to carry out the reaction at 0° C. for about 3 to 4 hours. Note that by adjusting the temperature and time of such heat treatment, the hardness of the carrier can be appropriately set depending on the molding and use. Sufficiently dried sepiolite can be easily crushed and granulated, and is the most preferred carrier for obtaining particulate immobilized products.

It is usually suitable to immobilize a peptide-containing substance onto such sepiolite using a coupling agent. For example, when immobilizing enzymes, silane coupling agents [e.g., r-aminopropyltriethoxy 7y
It is preferable to use Sr-chloropyltrimethoxysilane, vinyltriethoxysilane, r-glycidoxyglobiltrimethoxysilane, N-β-(aminoethyl)-1-aminopropyltrimethoxysilane, etc. ,
Preferably, γ-aminopropyltriethoxysilane is used. For immobilization using these, for example, sepiolite is added to these aqueous solutions or kept in contact with them, the silane coupling agent is introduced by reacting with the hydroxyl group in sepiolite, and then a bifunctional immobilization agent (for example, After treatment with an aqueous solution of glutaraldehyde, etc.), the enzyme is immobilized by chemical bonding with the silane coupling agent residue in Seviolite by contacting with an aqueous solution of the desired enzyme. However, in some cases, it is also possible to directly immobilize without using a coupling agent, such as in the so-called cyanogen bromide activation method.

The peptide-containing substance immobilized product obtained by K in this way is
This activity is due to the abundant hydroxyl groups of sepiolite and its large specific surface area (230 to 26 G'/,
) K-dipeptide-containing compounds have good Mg effect and are thought to be able to produce T-electrification with a strong Bi-tsuta aroma. This is thought to be due to the fact that the immobilization and the action of the immobilized substance on the substrate etc. are carried out efficiently.

(e) Examples This invention will be explained below with reference to Examples.

(Introduction of Silane Coupling Agent) The Seviolite lump was heat-treated at about 450° C. for 180 minutes, sufficiently dried, and crushed to form mesh ω~1200 particles. This granular sepiolite 50f was purified by hydrochloric acid.
5 volumes of r-7 minoprobyltriethoxysilane (silane coupling agent) adjusted to H3,5'Jt solution 1
After the pH became 3 or less, r-aminopropyltriethoxysilane was further added to adjust the pH to 3 to 4, and the mixture was heated under reflux in an oil path at 85°C for about 2 hours. Stir gently.

The granular Seviolite after the reaction was filtered through a membrane filter, washed with 500 (1 wt) water and 200 wt acetone, and dried at 120° C. for 2 hours.

(Immobilization of Enzyme) A glutaraldehyde solution 100-100% was prepared by adding sodium phosphate buffer to 25 volumes of glutaraldehyde 10-100% to the aminosilanized Seviolite obtained above.
and adjust the pH to about 6 using Magnetech Star 2.
The mixture was stirred for 910 minutes, then degassed for 30 minutes, and stirred in a water bath incubator (30° C.) for 20 minutes. Thereafter, the mixture was washed with a sodium phosphate buffer solution of pH 7, 5000-mL, and left under reduced pressure for half a day.

The aminosilanized Seviolite 100 cape treated with glutaraldehyde thus obtained was added to a solution of β-galactosidase (40q) in sodium phosphate buffer (PH
7,0) When the immobilization reaction is carried out by adding 5sg of K and shaking in a hot water bath (30℃) for 5 hours, β
- The immobilized enzyme of the present invention (in the form of beads) was obtained by immobilizing galactosidase on Seviolite.

To evaluate the activity of the immobilized enzyme thus obtained, see below! The amount of glucose produced under the own conditions was measured using a glucose meter.

(Conditions) Substrate: 157-lactose sodium phosphate buffer -20
0 mg, pH 6,6 Reaction temperature = 30°C Amount of immobilized enzyme used: 100 caps The results are shown in Figure 1. 0 Thus, the above immobilized enzyme acts on lactose to hydrolyze it into glucose and galactose. It is found that it has good enzyme activity.

B) Effects of the Invention As mentioned above, the immobilized product of the present invention is one in which a peptide-containing substance is directly immobilized without preparing the carrier itself in advance by chemical treatment, etc. as in the past. Therefore, it can be manufactured more easily. Furthermore, the carrier itself is a natural mineral and is much safer than conventional carriers. It can be used in both summer and summer areas. Moreover, this carrier is generally available at a very low cost, and combined with a simple manufacturing method, it has the advantage of producing a very cheap immobilized product.

[Brief explanation of drawings]

Figure 1 L1 is a graph illustrating the activity of the β-galactosidase immobilized enzyme of one embodiment of the present invention.

Claims (2)

[Claims] (1) A peptide-containing substance immobilized product comprising a functional peptide-containing substance such as an enzyme, antigen, or antibody immobilized on a carrier by chemical bonding, characterized in that sepiolite is used as the carrier. (2) The immobilized product according to claim 1, wherein the carrier is made of sepiolite that has been dried by heating.