JPS5853914B2 - Enzyme immobilization method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for obtaining an immobilized enzyme that can be used continuously and repeatedly in order to effectively utilize enzymes in enzyme-utilizing reactions. do.

In recent years, with the progress of enzyme immobilization technology, attempts have been made to make effective industrial use of the specific catalytic action of enzymes, such as sensors using immobilized enzymes or drug production.

The enzyme immobilization methods used in this process include the entrapment method in which the enzymes are immobilized in a polymer matrix, the carrier binding method in which the enzymes are directly chemically bonded to the immobilization carrier, and the crosslinking method in which the enzymes are cross-linked and immobilized to each other. There is.

Specifically, various organic polymers or inorganic substances such as glass beads and carbon are used as carriers, and the enzyme is immobilized on these carriers using the above immobilization method.

In the carrier binding method, the enzyme is directly bound to the carrier, so the enzyme does not fall off during use, but the various reactions and operations during immobilization are complicated, and the types of carriers that can be applied are limited.

In the entrapment method, the enzyme is immobilized by a method such as confinement in a three-dimensional resin matrix.

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

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

On the other hand, in the cross-linking method, a cross-linking reaction is carried out between the enzymes or between the enzyme and the carrier using an immobilizing reagent such as glutaraldehyde, thereby immobilizing the enzyme in solubility.

Specifically, the surface of the immobilization carrier to be used is coated with an enzyme solution by coating or dipping, dried if necessary, and then an immobilization reagent solution such as glutaraldehyde is added to perform a crosslinking reaction. , to crosslink and insolubilize the enzyme.

Generally, an aqueous solution of glutaraldehyde is used, but if the concentration of the immobilizing reagent is high, the crosslinking reaction will proceed rapidly and the activity of the enzyme will be lost.

On the other hand, if the concentration of the immobilized reagent is low, the reaction rate is slow, and during this time unreacted enzyme is dissolved in the added immobilized reagent solution.

In immobilization, the concentration of the enzyme needs to be above a certain level, and a decrease in the enzyme concentration due to the added immobilization reagent solution is very disadvantageous for the reaction to proceed.

As is clear from these points, it is almost impossible to uniformly cover the surface of the carrier with a film made of immobilized (insolubilized) enzyme using this method.

That is, when performing the immobilization reaction (crosslinking reaction), the above-mentioned problems are unavoidable because the immobilization reagent is applied in a solution state, that is, the immobilization reagent is supplied from the liquid phase.

Therefore, the present inventors conducted repeated studies to solve the problems of various immobilization methods, and as a result, discovered an immobilization method in which an immobilization reagent that crosslinks with the enzyme is supplied from the gas phase.

Its feature is that when an immobilization reagent such as glutaraldehyde is applied to cross-link and immobilize the enzyme and the carrier, or between the enzymes, the immobilization reaction is carried out while the immobilization reagent is supplied from the gas phase.

That is, the enzyme can be effectively immobilized by once converting the immobilization reagent into a gaseous state, coating it with an enzyme solution, and supplying it to the surface of the carrier, which has been further dried if necessary.

Therefore, we further investigated this immobilization method from the perspective of improving the activity of the immobilized enzyme, and found that by performing the immobilization reaction in a non-oxygen atmosphere, we could obtain an immobilized enzyme with excellent enzyme activity. It turned out that it can be done.

That is, under an inert gas atmosphere such as argon or nitrogen,
Alternatively, by supplying the immobilization reagent from the gas phase under reduced pressure to advance the immobilization, it was possible to significantly reduce the decrease in enzyme activity associated with immobilization.

The reason for this effect is not clear, but it is thought that oxygen in the reaction system is involved in some way in the immobilization reaction that affects enzyme activity. It is thought that the above effects can be obtained under conditions where the oxygen concentration is reduced.

Furthermore, in the immobilization method of the present invention, the concentration of the immobilization reagent involved in the immobilization reaction can be easily controlled by its vapor pressure, so that the immobilization reaction, which has a significant effect on subtle changes in the activity of the enzyme, can be easily controlled. You can easily adjust the progress rate.

As described above, in the enzyme immobilization method of the present invention, it is possible to obtain an immobilized enzyme with high enzyme activity by combining two reaction conditions: a non-oxygen atmosphere and supply of the immobilization reagent from the gas phase. It is possible.

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

FIG. 1 is a cross-sectional membrane diagram showing an example of an enzyme electrode for measuring substrate concentration in which an enzyme is immobilized on the surface of a Nesa glass electrode by the method of the present invention.

In the figure, 1 is an immobilized enzyme membrane consisting of alcohol dehydrogenase.
2 is a Nesa film, and 3 is a glass layer.

The method for manufacturing this enzyme electrode is as follows.

First, a phosphate buffer solution in which alcohol dehydrogenase was dissolved at a ratio of 100 m9/- was applied and developed on the surface of the aforementioned Nesa glass electrode (diameter 10 m, thickness 1 mπ).

Then, after being slightly dried, it was heated to 60°C at 25°C in glutaraldehyde vapor under various atmospheric conditions shown below.
The immobilization reaction was allowed to occur for minutes.

After the reaction was completed, it was washed with phosphate buffer.

The immobilized enzyme membrane thus obtained had sufficient adhesion strength.

Regarding the reaction atmosphere conditions, A is the electrode obtained under reduced pressure (30 imHg or less), B is the case with argon gas, C is the case with nitrogen gas, D is the case with air, and E is the case with oxygen gas.

Ethanol concentration was measured using the alcohol dehydrogenase immobilized electrode in the following manner.

Using an H-type cell, the electrode described above was attached to a resin electrode holder and immersed in a phosphate buffer solution of pH 7.0 containing 1×10 −3 mol/l nicotinamide adenine dinucleotide as a coenzyme.

Next, after setting the electrode potential to a sufficient oxidation potential of the coenzyme using a potentiostat, ethanol was injected to reach a predetermined concentration, and the oxidation current value of the coenzyme reduced by the enzyme reaction was measured.

Figure 2 shows the increase in current value as the ethanol concentration changes.

As is clear from the figure, electrode A according to the invention, compared to electrode H obtained under air or enzyme atmosphere,
The increase in current in B and C is large, indicating that the activity of alcohol dehydrogenase immobilized on the electrode is high.

Such an activity-improving effect is not limited to the above-mentioned alcohol dehydrogenase.

Similar effects were obtained when the immobilization method of the present invention was used for various enzymes.

In addition to the glutaraldehyde shown in the examples, examples of immobilization reagents that can be used in the immobilization method of the present invention include aldehydes such as 2-oxyacibaldehyde, crotonaldehyde, acrolein, glyoxal, propionaldehyde, and paraformaldehyde. Alternatively, any immobilizing reagent that can be supplied from the gas phase, including aldehyde polymers, can be used.

As described above, according to the enzyme immobilization method of the present invention, an immobilized enzyme having high enzyme activity can be easily obtained.

[Brief explanation of drawings]

FIG. 1 is a schematic cross-sectional view showing a Nesa glass electrode and an immobilized enzyme membrane covering it, and FIG. 2 shows the relationship between ethanol concentration and current increase amount measured using various enzyme electrodes. 1: Immobilized enzyme membrane, lath layer. 2...Nesa membrane, 3...Ga

Claims (1)

[Scope of Claims] 1 An enzyme immobilization method in which an enzyme is immobilized with an immobilization reagent that undergoes a crosslinking reaction with the enzyme, wherein the immobilization reagent is supplied from a gas phase under reduced pressure or an inert gas atmosphere. An enzyme immobilization method characterized by carrying out a chemical reaction. 2. The enzyme immobilization method according to claim 1, wherein the immobilization reagent is selected from aldehydes and aldehyde polymers.