JPS61149086A - Production of enzyme immobilized membrane - Google Patents

Abstract

PURPOSE:To obtain the titled membrane having uniform membrane thickness simply and surely even if the surface of solid surface is large, by sticking the flat solid surface fast to a thin membrane of enzyme solution spread on a frame, and sticking the solid surface having formed the enzyme membrane fast to a thin membrane of a solution of a polyfunctional reagent spread on a frame. CONSTITUTION:The structure 1 is immersed in a solution of an enzyme or it and protein, and slowly pulled up to form the thin membrane 2. The surface of the flat solid 3 such as plate glass, etc., is pressed against the thin membrane 2 and stuck fast to it. Then, the surface of the solid having formed the enzyme membrane is pressed against a thin membrane of a polyfunctional reagent such as glutaraldehyde, etc. or it and a surface active agent spread on another frame structure, stuck fast to it, and a crosslinking reaction is carried out, to give an enzyme immobilized membrane. The enzyme immobilized membrane having uniform membrane thickness simply and surely even if the area of the solid surface is large is formed on the surface of the solid.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing an enzyme-immobilized membrane in which the enzyme-immobilized membrane is formed in close contact with a solid surface.

(Prior art and problems) Conventionally, as a method for forming an immobilized enzyme film on a solid surface by crosslinking and insolubilizing an enzyme with a suitable protein using a polyfunctional reagent, an aqueous solution of enzyme and protein k = functional A method is known in which a mixture of chemical reagents is spread on a solid surface using an all-glass rod (for example, in Analytical Chemistry).
emistry) Vol. 49, No. 6, pp. 795-798 (1
977)).

However, with the enzyme-immobilized membrane obtained by this method, the development of the mixture must be completed within a few seconds, and when the surface area of this method becomes large, it is difficult to form a membrane with a uniform thickness. .

(Objective of the Invention) An object of the present invention is to provide a simple and reliable method for producing an enzyme-immobilized membrane, which solves all of the conventional drawbacks.

(Structure of the Invention) The present invention involves forming an enzyme film on the solid surface by pressing a planar solid surface in parallel to a thin film of an enzyme or an enzyme and protein solution stretched over a frame-like structure. and a step of pressing the solid surface on which the enzyme film is formed parallel to a thin film of a polyfunctional reagent or a solution of a polyfunctional reagent and a surfactant stretched on a frame-like structure to bring it into close contact. An object of the present invention is to provide a method for producing an enzyme-immobilized membrane characterized by the following.

(Function) The enzymes targeted by the present invention are not particularly limited in type; examples of enzymes include hydrolytic enzymes (amylase, grotease, pectinase, invertase, etc.), oxidoreductases (glucose oxidase, catalase, etc.)
% isomerase (glucose isomerase, etc.).

The enzyme or solution of enzyme and protein in the present invention may be appropriately determined so that the concentration is generally 15 to 40% by weight and within a range that allows the frame-shaped structure to be formed.

The polyfunctional reagent used in the present invention may be one commonly used as an enzyme or a crosslinking agent between an enzyme and a protein, such as glutaraldehyde, hexamethylene diinthiocyanate, and the like.

If it is difficult to apply a thin film on a frame-like structure with a solution containing only a polyfunctional reagent, it is usually necessary to apply a thin film to the frame-like structure so that the concentration is 0.01 to 0.1% by weight. It is possible to add a surfactant to the polyfunctional reagent solution to the extent that a thin JI can be obtained. The surfactants used here include anionic surfactants (sodium dodecyl sulfate, sodium dodecyl sulfonate, etc.) and cationic surfactants (dodecyltrimethylammonium chloride.

dodecylpyridinium bromide, etc.), amphoteric surfactants (balmitoyl lysolecithin, dodecyl-N-betaine, etc.), nonionic surfactants (Triton)
(-too, lauryldimethylamine oxide, etc.)
Examples include surfactants with a steroid skeleton (sodium cholate, sodium deoxycholate, etc.), but it is preferable to use nonionic surfactants that have a mild effect on enzymes.

The planar solid surface in the present invention may be one having appropriate water wettability (glass, 5isljJ4, etc.), or one surface may be coated with a wood-philic primer such as (3-aminopropyl)triethoxysilane. It may also be chemically modified.

・The flatness of the solid surface is not strictly required;
There are some unevenness, but there is no major problem. Furthermore, there are no restrictions on the shape of the solid surface.

In the present invention, the frame-shaped structure on which a thin film of enzyme or enzyme and protein is stretched has appropriate water wettability.
For example, a frame having a shape similar to the above-mentioned planar solid surface may be prepared using a glass rod having a diameter of approximately 5 B, and this may be used as a frame-like structure.

Methods for applying a thin film of an enzyme or a solution of an enzyme and a protein to such a frame-like structure include immersing the frame-like structure in the above-mentioned solution of the enzyme or an enzyme and a tannomic substance, and then gently pulling it up; A method may be adopted in which an enzyme or a solution of an enzyme and a protein is applied to a frame-shaped structure, and the solution is spread on the frame-shaped structure using a glass rod or the like.

When the above-mentioned planar solid surface is pressed parallel to the thin film of the enzyme or enzyme-protein solution stretched over the frame-shaped structure as described above, the thin film adheres uniformly to the planar solid surface. Then, an enzyme film is formed on the solid surface.A uniform thin film of the enzyme and protein solution is formed in close contact with the solid surface.

Next, a thin film of a polyfunctional reagent or a solution of a polyfunctional reagent and a surfactant is spread on a frame-like structure in accordance with the above-mentioned method of spreading an enzyme or a solution of an enzyme and a protein on a frame-like structure. Next, the solid surface held in a thin wire tube containing an enzyme or an enzyme and protein solution is pressed in parallel to bring the solid surface and the thin film into close contact. After this operation, the crosslinking and reaction of the multifunctional reagent is completed under appropriate conditions. This is 5K
By doing so, the enzyme-immobilized membrane of the present purpose can be uniformly formed in close contact with the solid surface, and the thickness of the formed enzyme-immobilized membrane can be 5 to 10 μm.

(-Example) The present invention will be described below with reference to FIG. 1(a), ←) showing an example.

A circular frame 1 with a diameter of 6 cIIL was formed from a glass rod with a diameter of 5 mm, and this was immersed in an IM phosphate buffer solution containing Ott% glucose oxidase and 15% by weight of bovine serum albumin, pH 7.0. Gently remove the excess glucose oxidase and bovine serum albumin solution, and apply a thin film 2 of the glucose oxidase and bovine serum albumin solution to the frame.A circular glass plate 3 with a diameter of 5α is placed parallel to this thin film 2. Press the thin film 2
A thin solution of glucose oxidase and bovine serum albumin was poured onto the T-glass plate 3 so as to tightly cover it.
t-m1 diagram (formed as shown in -.Furthermore, another glass rod frame having the same shape as the above glass rod frame f: 15 weight -
glutarartehi)”, 0.1 weight it% (F)
Tri ton Hang it on the frame. A thin film of the above-mentioned glucose oxidase and bovine serum albumin solution was formed on this thin film, and a glass plate was pressed in parallel to bring it into close contact with the thin film to initiate a crosslinking reaction with glutaraldehyde. Melt. Crosslinking reaction t
After z minutes, the glass plate was thoroughly washed with distilled water and further immersed in a 0.1M glycine solution for m minutes to remove excess glutaraldehyde. In this way, the desired enzyme-immobilized membrane is formed on the glass plate. The thickness of the formed enzyme-immobilized film was about 7 μm and was uniform over the entire surface of the glass plate.

The glass plate on which the enzyme-immobilized membrane was formed was reacted with a reaction solution having the following composition at 25°C with slow stirring, and the activity of glucose oxidase in the enzyme-immobilized membrane was determined from the change in absorbance at a wavelength of 436 nm $PL. Measure.

Composition o 0.66 ml of O-dianisidine hydrochloride, oxygen saturated 0.1 M +77 acid buffer pH 7.0
----------・-25m lo 100mg/1nl
l D-glucose solution...-5fF1102
m, ji /ml peroxidase solution...
...0.1 According to the measurement results, the glucose oxidase in the enzyme-immobilized membrane had the activity of 17q6 of the original glucose oxidase.

(Effect of the invention) When the area becomes large, it is difficult to form an immobilized film with a uniform thickness, but with the deep manufacturing method of the present invention, even if the area of the solid surface is large, it is possible to easily and reliably form a uniform film. It is possible to form thick enzyme-immobilized films on solid surfaces.

[Brief explanation of drawings]

FIGS. 1(a) and 1(b) are diagrams showing one embodiment of the present invention. Figure 71 O)

Claims (1)

[Claims] A process of forming an enzyme film on the solid surface by pressing a planar solid surface into close contact with a thin film of an enzyme or an enzyme and protein solution stretched on a frame-shaped structure, and a process of forming an enzyme film on the solid surface; production of an enzyme-immobilized membrane, comprising the step of pressing a solid surface on which the enzyme membrane is formed onto a thin film of a solution of a polyfunctional reagent or a solution of a polyfunctional reagent and a surfactant, and bringing the enzyme membrane into close contact with the thin film. Method.