JPH06189762A - Carrier having immobilized enzyme, its production and enzyme sensor produced by using the carrier - Google Patents

Abstract

(57) [Summary] [Structure] An enzyme-immobilized carrier having an enzyme immobilized on β-chitin, a method for producing the same, and an enzyme electrode comprising the enzyme-immobilized carrier and an electrode for detecting a reaction of the enzyme. I had an enzyme sensor. [Effects] The enzyme-immobilized carrier of the present invention has high stability of enzyme activity, can be prepared into various shapes by simple operations, and can be used in various applications such as enzyme sensors and is extremely useful. It is a thing.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzyme-immobilized carrier, a method for producing the same, and an enzyme sensor using the same, and more specifically, enzyme immobilization to which enzyme activity is imparted by immobilizing the enzyme on β-chitin. And a method for producing the same, and an enzyme sensor using the same.

[0002]

2. Description of the Related Art In recent years, in various industrial fields including foods, by using an enzyme-immobilized carrier in which a water-soluble enzyme is immobilized on a water-insoluble polymer carrier, the enzyme reaction process can be carried out continuously. Research into methods for repeated use of enzymes is in progress. In addition, research on enzyme electrodes in which a membrane-shaped enzyme-immobilized carrier and various electrodes are combined has been actively conducted.

When an enzyme is immobilized on a carrier on an industrial scale, it is necessary that the immobilization operation be simple, the immobilized enzyme be kept stable for a long time, and the carrier used be porous and inexpensive. Is. When used for an enzyme electrode, it is necessary that the carrier has good substrate permeability and can be formed into a thin film.

However, the conventional carrier often requires treatment such as cross-linking when immobilizing the enzyme, and secondary enzyme deactivation due to heat or an organic solvent is unavoidable during the cross-linking. There was a flaw.

On the other hand, chitin is an abundant natural resource, and it has been proposed to utilize a molded product of chitin as a carrier for immobilizing an enzyme (Japanese Patent Publication No. 56-35, 5).
3-10150 and 61-1116686).

[0006] Conventionally, as a method for preparing a chitin molded body, a method has been known in which chitin separated from the exoskeletons of crustaceans and insects by treatment with hydrochloric acid and caustic soda is used (JP-A-61-53339). JP, 61-64256, 61-129005, 61-21230.
No. 2).

[0007]

However, the conventional chitin obtained from the exoskeletons of crustaceans and insects is an α-type chitin structurally (hereinafter referred to as α-chitin) and thus has a poor dope forming ability. The resulting carrier has the drawback of poor strength, etc. Moreover, when it is attempted to use a film-like carrier for use in enzyme electrodes, etc., it does not become a non-woven fabric and does not become a thin film, and it is put to practical use as an enzyme carrier. Has not reached.

[0008]

Under such circumstances, the inventors of the present invention have conducted diligent research to solve the above-mentioned problems, and found that among many chitin raw materials, β-form structurally obtained from squid shells. Chitin (hereinafter referred to as β-chitin)
Among them, β-chitin obtained from the squid soft shell belonging to the mollusc genus Tsudika has excellent properties as a carrier, and by immobilizing an enzyme on it, an enzyme carrier having high enzymatic activity can be easily obtained, and thin film formation is possible. It has been found that it can also be used as an enzyme electrode because it is possible to complete the present invention.

That is, the present invention provides an enzyme-immobilized carrier characterized by immobilizing an enzyme on β-chitin, a method for producing the same, and an enzyme sensor using the same.

As β-chitin in the present invention, for example, β-chitin isolated from squid shell can be used.

To separate β-chitin from squid carapace, the squid carapace is first crushed and then treated with caustic soda and hydrochloric acid to remove proteins and ash. Taking squid soft shell as a raw material, for example, crushed squid carapace is treated with caustic soda at a concentration of about 1N at a temperature of about 90 ° C for about 1 hour, and then hydrochloric acid at a concentration of about 0.1N is added. Use at room temperature for about 1
Perform time processing. If this treated product is dried, β-chitin can be obtained.

In order to obtain the enzyme-immobilized carrier of the present invention, sufficiently ground (preferably 16 mesh or less) β-chitin is suspended in water at an appropriate concentration such as 5 to 20% by weight, and then vigorously stirred. Alternatively, freeze-thaw treatment is performed. In this way, β-chitin absorbs water to increase its viscosity and finally forms a gel. This gel is evenly dispersed in water, and a predetermined amount of enzyme solution is added to this dispersion to make it even
-A chitin dispersion may be prepared, and further drying and molding may be performed.

The shape of the enzyme-immobilized carrier of the present invention is not particularly limited, and may be in the form of a film, particles or the like depending on its use. For example, the enzyme-containing β-obtained as described above
The chitin dispersion is poured onto a filter to remove water from the bottom, so-called papermaking is performed, and then dried to obtain a membrane-shaped enzyme-immobilized carrier.

The enzyme immobilized on β-chitin may be any enzyme as long as it has an enzymatic activity, and a plurality of enzymes can be combined and immobilized.

When the enzyme-immobilized carrier is produced by using α-chitin obtained from crabs instead of β-chitin, chitin gel is obtained even if the above-mentioned stirring or freeze-thawing treatment is carried out. Since a substance cannot be obtained, it is necessary to form chitin fiber once and use it to prepare an enzyme-immobilized carrier. Therefore, for example, when a membrane-shaped enzyme-immobilized carrier is produced from α-chitin, a non-woven fabric-like product is obtained, which is completely different from the membrane-shaped enzyme-immobilized carrier of the present invention, and has substrate permeability and The strength is also inferior.

The thus obtained membrane-shaped enzyme-immobilized carrier of the present invention and an electrode for detecting the reaction of the immobilized enzyme can be combined to be used as an enzyme electrode.
Here, the electrode for detecting the reaction of the enzyme is not particularly limited, but, for example, when the enzyme is β-D-glucose oxidase, an oxygen electrode, a hydrogen peroxide electrode or the like is used. By using an enzyme electrode in which a membrane-shaped enzyme-immobilized carrier is mounted on these electrodes, an enzyme sensor for quantifying glucose can be obtained.

[0017]

EXAMPLES Next, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

Example 1 (1) Feather mill (5 m /
m, screen bath), and the crushed product is 1N NaO
Put in H solution, heat at 90 ℃ for 1 hour, wash with water, then 0.1
Immerse in N HCl solution at room temperature for 1 hour. Then, after washing with water, the mixture was heated with a 1N NaOH solution at 90 ° C. for 1 hour, washed with water, and dried in an oven at 50 ° C. for 5 hours to obtain 100 g of β-chitin.

(2) Next, the β-chitin was ground, and water was added so that the dry ground product had a concentration of 0.2% by weight.
Stir vigorously to increase viscosity and form a pasty gel. To 25 ml of the gel thus obtained, an aqueous solution of β-D-glucose oxidase (manufactured by Sigma) (2 mg / m 2
l) 25 ml was added, and this solution was subjected to a conventional water-drop falling papermaking. This is air dried at 4 ° C for 15 hours, β-D
-Glucose oxidase-immobilized membrane was obtained. The absorbance at 280 nm of the liquid before and after paper making was measured, and the amount of enzyme adsorbed on the membrane was measured. As a result, 86% of the enzyme was adsorbed.

Example 2 The β-D-glucose oxidase-immobilized membrane prepared in Example 1 was set on a cathode having a diameter of 3 mm of an oxygen electrode,
An enzyme electrode was constructed. This enzyme electrode was set in the flow cell and the flow rate was 1.16 ml / min at 25 ° C and 50 mM phosphate buffer solution.
(pH7.4) was passed through, and 20 μl of 100 mM glucose solution was injected into this channel with a microsyringe. The response of the sensor at that time was examined as a change in the potential generated across the 22 kΩ resistance placed in the circuit. It was For comparison, a Millipore membrane filter TYPE HA (1.0μ
m) was immersed in a β-D-glucose oxidase 1.5 mg / ml solution and adsorbed and fixed, and a membrane was similarly set on the electrode for measurement. The results are shown in FIG.

As a result, the activity of the membrane filter adsorbing β-D-glucose oxidase was greatly reduced in 20 to 30 minutes, whereas the activity of the membrane immobilized with β-D-glucose oxidase of the present invention was not observed at all. I couldn't do it. In addition, if the liquid flow is continued as it is, β-D-
The glucose oxidase-immobilized membrane maintained its activity even after continuous operation for 3 days.

Furthermore, when this β-D-glucose oxidase-immobilized membrane was stored in a freezer at -25 ° C. for 1 week and the enzyme electrode was similarly assembled, the same amount of response to the glucose solution was obtained. , It was also found to be resistant to freezing.

Example 3 Water was added so that the dry ground product of β-chitin obtained in Example 1 had a concentration of 0.2% by weight, and the mixture was vigorously stirred to form a paste-like gel. 10 ml of gel thus obtained
To the solution, 10 ml of an aqueous solution of β-D-glucose oxidase (manufactured by Sigma) (10 mg / ml) was added, and this solution was subjected to a conventional water-drop falling papermaking. This was air-dried at 4 ° C for 5 hours, and the obtained membrane was stirred in a sufficient amount of distilled water for 1 hour to wash off the enzyme which was incompletely adsorbed, and again at 4 ° C for 5 hours.
Air drying was carried out for an hour to obtain a β-D-glucose oxidase-immobilized membrane.

This membrane was set on the cathode of an oxygen electrode to form an enzyme electrode. This was put in 20 ml of 50 mM phosphate buffer (pH 6.0) saturated with air kept at 30 ° C., and after the output current became constant, 100 mM glucose became a final concentration of 0.125-4.0 mM. In addition, the change in output was examined.

The results are shown in FIG. As a result, at any concentration, the output current decreased immediately after the sample injection, and became stable within 50 seconds. The amount of change at this time is plotted against the glucose concentration in FIG. A linear relationship was obtained at a glucose concentration of 0.125-2.0 mM.

Example 4 The β-D-glucose oxidase-immobilized membrane prepared in Example 3 was treated with a hydrogen peroxide analyzer of the flow injection analysis type (FAM-1A, manufactured by Toa Denpa Kogyo KK). Mount on the electrode and continuously add 100 mM phosphate buffer (pH 6.0) containing 0.1 M KCl at 1.0 ml / min, 35
The solution was delivered at 0 ° C., and 10 and 100 mM glucose solutions were alternately injected to measure.

The results are shown in FIG. From this experiment, no decrease in response was observed even after continuous use for 24 hours.

[0028]

INDUSTRIAL APPLICABILITY The enzyme-immobilized carrier of the present invention has a high stability of enzyme activity, can be prepared into any shape by simple operation, and can be used in various applications such as enzyme sensors, It is useful.

[Brief description of drawings]

FIG. 1 is a drawing showing the results of potential changes of a β-D-glucose oxidase-immobilized membrane of the present invention and a β-D-glucose oxidase-adsorbing membrane filter in Example 2.

FIG. 2 is a drawing showing changes in the amount of output current with time of the enzyme sensor of the present invention in Example 3.

FIG. 3 is a diagram showing the relationship between glucose concentration and current change amount in Example 3.

FIG. 4 is a view showing a change in response of the enzyme sensor of the present invention due to continuous glucose administration in Example 4.

Claims (3)

[Claims] 1. An enzyme-immobilized carrier comprising an enzyme immobilized on β-chitin. 2. The method for producing an enzyme-immobilized carrier according to claim 1, which comprises dissolving the enzyme solution in the β-chitin dispersion and then molding the solution. 3. An enzyme sensor using an enzyme electrode comprising the enzyme-immobilized carrier according to claim 1 and an electrode for detecting a reaction of the enzyme.