JPS5913188B2 - Manufacturing method of enzyme immobilized membrane - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION An object of the present invention is to obtain an immobilized enzyme that can be used continuously and repeatedly in order to effectively utilize enzymes in enzyme-utilizing reactions.

In recent years, with the progress of enzyme immobilization technology, attempts have been made to effectively utilize the specific catalytic action of enzymes in industrial applications such as sensors and drug production using immobilized enzymes.

As one of the enzyme-immobilized bodies used in this, enzyme-immobilized membranes, such as those in which an enzyme is immobilized on a membrane, or those in which an enzyme is immobilized and formed into a membrane, are known.

In this case, the enzyme is integrally immobilized by a method such as an entrapping immobilization method in a polymer matrix or a carrier bonding method by direct chemical bonding to a carrier.

Specifically, an organic polymer such as a resin or cellulose is used as an immobilization carrier (retainer), and the enzyme is immobilized onto this carrier using the above method.

For example, in the carrier binding method, a functional group is introduced into the membrane in advance by chemical modification or the like, and then a reaction is performed with an enzyme to bond the functional group.

This method directly binds the enzyme and the carrier membrane, so
Although the enzyme does not fall off, the reaction during immobilization,
If the operation is complicated, usable carriers are limited.

In addition, in the comprehensive method, for example, 3
Enzyme immobilization membranes can be created by trapping the enzyme in a dimensional resin matrix, or by dissolving the resin together with the enzyme in a solvent, spreading it on soil such as a glass plate, and then removing the solvent and immobilizing it. can be obtained.

Since this method does not involve binding the enzyme to a carrier, there is little reduction in enzyme activity due to immobilization.

However, on the other hand, desorption of enzymes due to continuous and repeated use is unavoidable.

Therefore, the present inventors have repeatedly studied various enzyme-immobilized membranes in order to solve the problems, and as a result, have discovered an excellent method for producing enzyme-immobilized membranes.

In the method for producing an enzyme-immobilized membrane of the present invention, a membrane having pores is immersed in a solution containing an enzyme or a protein other than the enzyme, such as albumin, and then the enzyme or enzyme is soaked in the vapor of an immobilization reagent. This method is characterized by carrying out a protein immobilization reaction with a membrane, and can insolublely immobilize an enzyme or an enzyme and a protein other than the enzyme on the surface and within the pores of the membrane.

Figure 1 shows the enzyme-immobilized membrane obtained by the method of the present invention.
] is shown in the cross-sectional view of the membrane.

In the figure, 1 is a membrane having pores used as a carrier, 2 is an enzyme layer immobilized in the pores of the membrane 1, and 3 is an enzyme layer immobilized on the membrane surface.

The enzyme layers 2 and 3 are formed as immobilized layers by crosslinking and insolubilizing the enzymes with an immobilizing reagent such as glutaraldehyde, and are integrated with the carrier membrane to form an enzyme immobilized membrane.

Therefore, it is possible to obtain an immobilized enzyme membrane that is extremely thin and has excellent mechanical strength, and is not affected by the material of the membrane used as a carrier.

In addition, since the structure makes maximum use of the surface and pores of the membrane, it also has the advantage of being able to immobilize a large amount of enzyme compared to conventional methods.

An enzyme-immobilized membrane having the above-mentioned excellent properties can be produced as follows.

First, a membrane having pores used as a carrier is immersed in an enzyme solution to perform an impregnation treatment.

When using a water-repellent membrane, it is subjected to hydrophilic treatment with alcohol, acetone, etc. in advance.

After covering the inside of the pores and the membrane surface with the enzyme solution, dry as appropriate.
Next, the enzymes are cross-linked using an immobilization reagent such as glutaraldehyde to immobilize them insoluble.

During this immobilization reaction, the immobilization reaction (crosslinking reaction) is allowed to proceed while the enzyme-covered membrane is placed in the vapor of the immobilization reagent.

Conventionally, in cross-linking immobilization of enzymes using immobilization reagents such as glutaraldehyde, a method has been used in which the immobilization reagent is applied in a solution state.

However, with this method of supplying the immobilized reagent from the liquid phase, it is difficult to control the progress of the crosslinking reaction, which has a large effect on enzyme activity, and unreacted enzyme
There are problems such as dissolution in the added immobilization reagent solution.

As is clear from these points, it is extremely impossible to cover the surface and pores of a membrane with a uniform layer of immobilized enzyme using conventional methods.

In the production method of the present invention, the immobilization reagent is once made into a gaseous state, and a carrier film covered with an enzyme solution is placed in this vapor to supply the immobilization reagent from the gas phase to perform the immobilization reaction. It is.

It has been found that by this method, it is possible to easily obtain an enzyme-immobilized membrane having a uniform immobilized enzyme layer on the surface and within the pores of the carrier membrane, contrary to expectations.

The method of the invention has the following features.

First, since the concentration of the immobilization reagent involved in the immobilization reaction can be controlled by its vapor pressure, the degree of progress of the immobilization reaction can be easily controlled.

Furthermore, by reacting in the immobilization reagent vapor, it is possible to immobilize the enzyme in the pre-coated state without changing the enzyme concentration on the surface and in the pores of the membrane used as a carrier.

Therefore, the adhesion between the membrane used as a carrier and the immobilized enzyme layer is uniform and good.

Therefore, an enzyme-immobilized membrane having excellent properties can be obtained regardless of the surface shape and pore shape of the membrane used as a carrier.

Hereinafter, the present invention will be explained with reference to examples thereof.

A porous polycarbonate membrane (film thickness 8 μm) was used as the membrane as a carrier.
m, pore diameter 10 μm, pore density I x 105 pores/i), and glucose oxidase is used as the enzyme.

The membrane is immersed in an aqueous solution in which this enzyme is dissolved together with albumin, and the membrane surface and pores are covered with the enzyme solution.

Then, after a little drying, in glutaraldehyde vapor,
By performing the immobilization reaction at 25° C. for 60 to 90 minutes, an enzyme-immobilized membrane that is thin and has sufficient strength can be obtained.

This membrane was placed over the enzyme-permeable membrane of a galvanic cell enzyme sensor, and after it was immersed in a phosphate buffer solution of pH 5.6 and the current value became constant, glucose was added and the accompanying enzyme The reduction in reduction current was measured.

The results are shown in FIG. In the figure, A shows the case where the glucose oxidase-immobilized membrane according to the present invention is used.

For comparison, as an enzyme-immobilized membrane using the conventional encapsulation method,
A benzene solution of polystyrene and glucose oxidase was developed on a glass plate, and then the solvent was removed to prepare a glucose oxidase-immobilized membrane, which was subjected to the same measurements as described above.

This is indicated by B. When glucose is added at time 00 on the time axis in the figure and the concentration is set to 2X10-' mol/l-, the enzyme-immobilized membrane according to the present invention has both the time required for the current value to become constant and the amount of change. is better.

FIG. 3 shows the relationship between glucose concentration and current change when the enzyme-immobilized membrane according to the present invention is used.

As described above, substrate concentration can be measured quickly and with high sensitivity using the enzyme-immobilized membrane obtained by the method of the present invention.

Furthermore, the enzyme-immobilized membrane of the present invention exhibited excellent properties with little performance deterioration even after repeated or continuous use.

The immobilization reagent that can be used in the present invention is not limited to the glutaraldehyde described in Examples.

2-oxyacibaldehyde, crotonaldehyde, acrolein, glyoxal, propionaldehyde,
Any immobilizing reagent that can be supplied in a gas phase can be used, including aldehydes such as paraformaldehyde or aldehyde polymers.

In addition, by immobilizing the enzyme with a protein other than the enzyme, such as albumin or polylysine,
Immobilization (insolubilization) becomes easier, and a decrease in enzyme activity due to immobilization can be reduced.

[Brief explanation of the drawing]

Figure 1 is a cross-sectional schematic diagram showing the relationship between the carrier membrane of the enzyme-immobilized membrane and the immobilized enzyme layer, and Figure 2 is a diagram showing the relationship between current changes and time of a galvanic cell type enzyme sensor using the enzyme-immobilized membrane. , FIG. 3 is a diagram showing the relationship between glucose concentration and current change. 1... Membrane with pores, 2, 3... Enzyme layer.

Claims (1)

[Claims] 1. After a membrane having pores is immersed in a solution containing an enzyme or an enzyme and a protein other than the enzyme, an immobilization reaction between the enzyme or the enzyme and the protein is carried out in the vapor of an immobilization reagent. 1. A method for producing an enzyme-immobilized membrane, comprising: 2. The method for producing an enzyme-immobilized membrane according to claim 1, wherein the immobilization reagent is an aldehyde or a polymer of aldehyde.