JPH06181763A - Immobilized enzyme membrane - Google Patents

Abstract

PURPOSE:To obtain an immobilized enzyme membrane having high enzyme- including property and high long-term storage stability. CONSTITUTION:An enzyme is immobilized in a membrane consisting essentially of an unmodified silk fibroin in a silk yarn glandular cell derived from an insect of Lepidoptera.

Description

Detailed Description of the Invention

[0001]

This invention relates to an immobilized enzyme membrane,
More specifically, the present invention relates to an immobilized enzyme membrane suitable for measuring the concentration of a substance to be measured in a test solution while being attached to the surface of a base electrode.

[0002]

2. Description of the Related Art Conventionally, an enzyme has been used to detect a desired organic polymer in a test solution with high accuracy, focusing on the characteristic that it has a high selectivity for an organic polymer such as a protein. Proposed to be used. In this case,
If the enzyme is used as it is, it becomes extremely difficult to retain the enzyme and the detection accuracy of the organic polymer is deteriorated.Therefore, it is common to use the enzyme immobilized on a carrier. is there.

As a method for immobilizing an enzyme on a carrier as described above, there has been proposed a method in which regenerated silk fibroin is used as a carrier, and the regenerated silk fibroin is solubilized, and then the desired enzyme is bound while salting out and precipitating. (See, for example, Japanese Patent Laid-Open No. 61-187790). Here, silk fibroin is used as a surgical suture material, paying attention to the fact that it is an insect biopolymer and has excellent biocompatibility. Moreover, since silk fibroin has various functional characteristics represented by excellent enzyme entrapment, it is expected to exhibit excellent characteristics as an enzyme-immobilized carrier. In particular, the silk protein produced by silkworm biosynthesis in silk cells, which is a typical example of lepidopteran insects that spit silk fibers in a broad sense, has a characteristic primary structure (sequence order of amino acids constituting silk protein). It has a very special secondary structure and higher order structure (three-dimensional structure defined by the interaction between the peptide groups of the peptide main chain which is the main body of silk protein). Silk protein is composed of crystalline parts with well-aligned molecules and non-crystalline parts with disordered molecular arrangement, and mechanical properties such as dyeability, hygroscopicity, and easiness of elongation are similar to those of the amorphous parts. It is known that mechanical properties such as hardness and strength depend on the properties of the crystal part. The non-crystalline portion contains many chemically active amino acids with long molecular side chains such as arginine, glutamic acid, lysine, and tyrosine, whereas the crystalline portion contains alanine and glycine regularly. Are arranged. Thus, due to the coexistence of the amorphous part and the crystalline part and the difference in the type and sequence of the amino acids that make up each part, silk protein has a characteristic not found in other biopolymers. It will show the nature. That is,
Immediately after the silk protein aqueous solution is evaporated to dryness, the silk solid matter dissolves in water, but the silk solid matter is only lightly soaked at room temperature in an aqueous solution of a poor solvent such as methanol or ethanol that has a denaturing effect on the silk protein. Will show the property of not easily dissolving in water. On the other hand, collagen, which is a biopolymer different from silk protein, is subjected to ultraviolet irradiation treatment, or cross-linking bonds are formed between molecules by adding a drug such as aldehyde, which is harmful to the human body, Since it can be insolubilized in water, it can be seen that the silk protein, which can achieve insolubilization in water, is excellent because the microstructure such as how molecules are gathered changes only by treatment with alcohol or the like, which does not adversely affect the human body. As is clear from the above,
Silk fibroin is rich in moldability and can be subjected to centrifugal treatment, heat treatment,
Insolubilization in water can be achieved without using a special reagent only by performing a simple physical treatment such as a solvent treatment. In addition, silk fibroin fibers can be easily dissolved by weakening hydrogen bonds between sample molecules by a neutral salt solution as compared with biopolymers such as collagen, chitin and chitosan, and pure silk protein aqueous solution can be obtained by dialysis treatment. It can be prepared and has an advantage that a transparent film can be obtained by spreading an aqueous silk protein solution on a film of polystyrene, polyethylene or the like and drying. Here, the sample film immediately after drying and solidifying the silk protein aqueous solution is soluble in water, but by treating for a short time with a solvent such as methanol, which is a poor solvent for silk protein, the molecular shape, It is suitable for use as a carrier for an enzyme, since the way it collects and the like changes and the molecular aggregating property increases, making it insoluble in water.

A method of immobilizing glucose oxidase (hereinafter abbreviated as GOD) by using regenerated silk fibroin as an enzyme immobilization membrane has been proposed (for example, Immobilization of Gluco).
se Oxidase with Bombyx mo
ri Silk Fibroin by only S
tretching Treatment and I
ts Application to Glucose
Sensor, Makoto Demuraet
al, Biotechnology and Bio
engineering, Vol. 33, pp. 5
98-603, January 1989).

[0005]

In any of the above methods, regenerated silk fibroin obtained by once dissolving cocoon thread, silk thread or silk by-product spun by silkworms in a neutral salt solution is used as a raw material. However, the molecular agglutination is not so excellent, and the stability when the enzyme reaction of the test substance is carried out after the carrier on which the enzyme is immobilized is stored for a long period of time cannot be improved so much. Specifically, the temperature stability and pH stability related to enzyme activity are improved compared to the case where biopolymer such as gelatin, chitin, chitosan is used as a carrier, and the storage stability for 6 months. Is also guaranteed.
However, it is empirically known that storage stability over 6 months becomes poor. In addition, when a biosensor obtained by immobilizing an enzyme using recycled silk fibroin as a carrier is put into a living body, the output of the biosensor is likely to decrease, and the biosensor obtained by immobilizing the enzyme on the carrier is practically used. It is a big obstacle to the realization.

Under these circumstances, it has been earnestly desired to develop an immobilized enzyme membrane which is excellent in enzyme encapsulation ability and has long-term storage stability without lowering enzyme activity.

[0007]

The present invention has been made in view of the above problems, and is excellent in long-term storage stability, and
It is an object of the present invention to provide a new immobilized enzyme membrane that can effectively prevent the enzyme activity from being lowered.

[0008]

[Means for Solving the Problems] In order to achieve the above-mentioned object, the immobilized enzyme membrane according to claim 1 comprises an enzyme in a membrane mainly composed of native silk fibroin in silk gland cells derived from Lepidoptera insects. It is fixed. Here, examples of Lepidoptera insects include domestic silkworms and wild silkworms. In addition, the amount of the enzyme that is comprehensively immobilized on the unmodified silk fibroin is 1 to 70%, preferably 20 to 40% with respect to the absolute dry weight of the unmodified silk fibroin. The enzyme to be immobilized is
Examples thereof include GOD, uricase, cholesterol oxidase, cholesterol esterase, lactate oxidase and the like.

[0009]

With the immobilized enzyme membrane of claim 1, the enzyme is immobilized on a membrane whose main component is native silk fibroin in silk gland cells derived from lepidopteran insects represented by silkworm. It is possible to provide an immobilized enzyme membrane having remarkably long-term storage stability and high output as compared with an immobilized enzyme membrane obtained by immobilizing an enzyme on a membrane composed of regenerated silk fibroin. More specifically, the unmodified silk fibroin aqueous solution for obtaining the film may be a dilute solution of 3% or less in absolute dry weight concentration, and is preferably a dilute solution of 1% or less. Then, an enzyme is added to the diluted solution, spread on a polystyrene film, a polyethylene film, or the like, and evaporated to dryness to obtain a silk film on which the enzyme is immobilized. In addition, the insolubilization treatment of the silk film obtained by evaporating the unmodified silk fibroin aqueous solution to dryness is 20 to 20.
80% methanol solution, preferably 40-60% methanol solution, 2-60 minutes, preferably 2-30
Immersion treatment may be performed for about a minute. However, most preferably, the immersion treatment may be performed at an alcohol concentration of 50% for 10 to 30 minutes. Here, the solvent that can be used for the insolubilization treatment of the silk film is not limited to methanol, but a poor solvent for silk proteins, such as ethanol, can be adopted in the same manner as methanol as long as it does not reduce the activity of the enzyme. .
Further, by changing the amount of enzyme added to the unmodified silk fibroin aqueous solution in the above film forming step, silk films having different enzyme entrapment amounts can be prepared. Furthermore, in the step of evaporating to dryness, silk membranes having different thicknesses can be obtained by changing the amount of the unmodified silk fibroin aqueous solution, the evaporation rate, and the concentration of the aqueous solution.

The present invention provides the selection of enzyme immobilization materials used for biosensors as part of the present inventor's research on the chemical structure and physicochemical properties of silk proteins and the development and research of new application technologies in the non-clothing field. Enzyme immobilization method,
As a raw material for the membrane, liquid silk fibroin (undenatured silk fibroin) in silk glands in silkworm glands, which is a representative of beneficial insects among lepidopteran insects, is used as a raw material for the membrane. The inventors have found that an immobilized enzyme membrane excellent in enzyme encapsulation and long-term storage stability can be prepared by using it, and completed the present invention.

[0011]

EXAMPLES The present invention will be described in detail below with reference to examples.

[0012]

Example 1 A posterior silk gland was taken out from the matured silkworm body of a mulberry plant of mulberry leaf, washed thoroughly with water, and then liquid unmodified silk fibroin in the silk gland was dispersed in distilled water to increase the concentration by blast drying. A 0.7% unmodified silk fibroin aqueous solution was prepared. 20 ml of this unmodified silk fibroin aqueous solution was added with 3.75 mg of GOD (manufactured by Toyobo Co., Ltd.).
Add it in a constant temperature bath at ℃, and carefully and gently stir it.
Left for hours. Then, the unmodified silk fibroin aqueous solution to which GOD is added is spread on a polystyrene plate,
Water was evaporated to dryness under an atmosphere of 65% RH to prepare an enzyme-containing silk transparent film. Further, it is immersed in a 50 wt% methanol aqueous solution for 5 minutes, dried at room temperature, and
An insolubilized membrane of unmodified silk fibroin having OD immobilized thereon was prepared.

On the other hand, as a comparative example, waxes, pigments and the like contained in the silkworm cocoon layer were removed by a Soxhlet extractor using an ethanolbenzene-based mixed solution (volume ratio 1: 2). Marcel soap 0.
Put in a mixed solution of 2% and 0.05% sodium carbonate,
Silk fibroin fiber was prepared by removing sericin in the outer layer of cocoon yarn by boiling at 98 ° C. for 30 minutes. The bath ratio was 1: 100. A scoured silk fibroin fiber was prepared by drying at 50-60 ° C for 24 hours. 55 scouring silk thread
After treated with an 8% lithium bromide solution at 8 ° C. for 30 minutes to dissolve it, dialyzed with pure water using a cellulose dialysis membrane to prepare a regenerated silk fibroin aqueous solution having a concentration of 0.7%. To 20 ml of this regenerated silk fibroin aqueous solution, 3.75 mg of GOD (manufactured by Toyobo Co., Ltd.) was added in a constant temperature bath at 20 ° C as in the case of the unmodified silk fibroin aqueous solution, and the mixture was carefully stirred gently and left for 2 hours. did.
Next, the regenerated silk fibroin aqueous solution to which GOD was added was spread on a polystyrene plate, and water was evaporated to dryness in an atmosphere of 20 ° C. and 65% RH to prepare a transparent silk film containing an enzyme. Furthermore, 5% in 50% by weight methanol aqueous solution
It was soaked for a minute and dried at a room temperature to prepare an insolubilized membrane of regenerated silk fibroin having GOD immobilized thereon.

The insolubilized membrane (immobilized enzyme membrane) of both silk fibroins prepared as described above was attached to the surface of the hydrogen peroxide electrode via the hydrogen peroxide selective permeable membrane, and glucose was immobilized on the immobilized enzyme membrane. A glucose solution having a known glucose concentration (150 mg / dl) was spotted with a diffusion limiting membrane for limiting diffusion permeation, and the maximum value of the time derivative of the output current output from the hydrogen peroxide electrode was measured. . Further, glucose solutions having different glucose concentrations were sequentially spotted and the same measurement was performed to obtain a dynamic range (upper limit of glucose concentration at which output linearity is not impaired, hereinafter abbreviated as DR). Further, the change rate Δ% of the output current after storing the immobilized enzyme membrane for 1 day was also obtained. The results obtained are shown in Table 1.
Shown in.

[0015]

[Table 1]

As is clear from the measurement results in Table 1, a larger output can be obtained and the storage stability is excellent (the rate of change in output current is small) when the immobilized enzyme membrane of the example is used. ). Regarding DR, it seems that the immobilized enzyme membrane of the comparative example is larger, but the output is large when the immobilized enzyme membrane of the example is used.
The DR can be easily increased by increasing the diffusion and transmission limiting effect of the diffusion limiting film. Therefore, the immobilized enzyme membranes of Examples had sufficiently large DR due to the large output.
It turns out that

Although the posterior silk gland is taken out from the matured silkworm body of the silkworm mulberry grown in mulberry leaf, the middle silk gland may be taken out instead of the posterior silk gland. Since a large amount of liquid silk protein is stored in the posterior silk gland, a large amount of undenatured silk fibroin can be obtained. However, in this case, after thoroughly washing the removed middle silk gland, the silk gland cells were carefully removed with tweezers, and the liquid silk protein obtained by removing the silk gland cells was placed in a predetermined amount of distilled water, Leave it quietly for a predetermined time at a predetermined temperature, remove the supernatant by decantation,
A predetermined amount of distilled water is added again, the supernatant is removed by the decantation method, and a predetermined amount of distilled water is added again, and after a lapse of a predetermined time, an unmodified silk fibroin solution from which silk sericin is removed can be obtained.

[0018]

Example 2 A method similar to that of Example 1 was used.
The liquid unmodified silk fibroin was dispersed in distilled water from the silk gland taken out from the body of Raea Pernyi) to prepare a 0.6% aqueous solution of unmodified persimmon silk fibroin. Then, GOD was incorporated in the same manner as in Example 1 to prepare an insolubilized persimmon silkworm fibroin membrane containing GOD.

On the other hand, as a comparative example, a regenerated silk fibroin was prepared by treating the tussah cocoon layer as follows. That is, sericin was removed by treatment with a 0.1% aqueous sodium peroxide solution in an amount 50 times the weight of the cocoon layer at 95 ° C. for 1 hour. Next, the cocoon layer from which sericin was removed was dissolved in 7M lithium thiocyanate at 35 ° C., and then dialyzed with pure water for 3 days using a cellulose membrane to remove inorganic ions and the like. Then, a 0.6% concentration of regenerated tussah silk fibroin aqueous solution was prepared by blast drying. Then, GOD was included in the same manner as in the above Comparative Example to prepare an insolubilized regenerated persimmon silk fibroin membrane containing GOD.

Using the insolubilized membrane (immobilized enzyme membrane) of both mulberry silk fibroins prepared as described above, the time differential value of the output current output from the hydrogen peroxide electrode was measured in the same manner as in Example 1. The maximum value was measured and the DR was obtained. The obtained results are shown in Table 2.

[0021]

[Table 2]

As is clear from the measurement results in Table 2, a larger output can be obtained and a larger DR can be obtained by using the immobilized enzyme membrane of the example. Since the output is increased when the immobilized enzyme membrane of the example is used, DR can be further increased easily by enhancing the diffusion permeation limiting effect of the diffusion limiting membrane. Therefore, it can be seen that the immobilized enzyme membranes of Examples can further increase the DR due to the large output.

[0023]

Example 3 In the same manner as in Example 1, liquid unmodified silk fibroin was dispersed in distilled water from the posterior silk gland of mature silkworm of domestic silkworm, and dried by blowing air to obtain a 0.6% unmodified silk fibroin aqueous solution. Prepared. Then, after forming the film in the same manner as in Example 1, the film was immersed in a 50% by weight aqueous methanol solution for 20 minutes for insolubilization treatment.

Using the insolubilized membrane (immobilized enzyme membrane) of unmodified silk fibroin prepared as described above, the same procedure as in Example 1 was carried out (except that 300 ml / dl of glucose aqueous solution was used). The maximum value of the time derivative of the output current output from the hydrogen peroxide electrode was measured over a long period of time, and long-term stability was examined. Specifically, the immobilized enzyme membrane was immersed in a phosphate buffer of pH 7.0 kept at 4 ° C. for a long period of time and stored, and then the glucose solution was measured as a test solution to measure the output current time. Long-term stability was evaluated by calculating the differential value. The measurement results are shown in Table 3.

[0025]

[Table 3]

As is clear from the above measurement results, 10
Even if stored for a month, no substantial decrease in output was observed,
It was possible to achieve remarkably excellent long-term storage stability as compared with a conventional immobilized enzyme membrane (using regenerated silk fibroin as a carrier), which has poor storage stability after 6 months. It should be noted that the output value increases with the lapse of storage days because the immobilized enzyme membrane is stored in a pulled state,
It is considered that this is because the film thickness becomes smaller as the number of storage days elapses.

[0027]

Example 4 A transparent silk fibroin aqueous solution derived from domestic silkworm used in Example 1 and a regenerated silk fibroin aqueous solution prepared by the method described in Example 1 were evaporated to dryness on a polyethylene membrane to obtain a transparent solution. The membrane is immersed in a 50% by weight methanol aqueous solution at 25 ° C for 2 minutes, and then at 20 ° C and 65%.
The insolubilized immobilized enzyme membrane was obtained by allowing to stand in a thermo-hygrostat of RH for 3 hours and naturally drying. And
Fourier transform infrared spectrophotometer (1700X, Perki
(manufactured by n-Elmer) was used to observe the absorption band of the amide V region in both immobilized enzyme membranes, whose molecular morphology can be evaluated relatively easily.

In the infrared absorption spectrum of the immobilized enzyme membrane using unmodified silk fibroin, in addition to the absorption at 698 cm ⁻¹ which is attributed to β-type molecular morphology after 2 minutes of immersion, unmodified untreated silk A wide absorption at 644 cm ^-1 peculiar to the random coil morphology peculiar to fibroin also appeared as a small absorption peak. On the other hand, in the infrared absorption spectrum of the immobilized enzyme membrane using the regenerated silk fibroin having a dipping treatment time of 2 minutes, only a major peak appears at 698 cm ⁻¹ , which belongs to β-type molecular form, and the random coil form is observed. The absorption based on has disappeared. These results mean that the random form contained in the immobilized enzyme membrane using regenerated silk fibroin is easily crystallized by the treatment with methanol. On the other hand, the immobilized enzyme membrane using undenatured silk fibroin has a dense surface structure with good molecular aggregating property, so it is difficult to be affected by methanol treatment, and random morphological regions are difficult to crystallize. The absorption peak at 644 cm ⁻¹ peculiar to the coil form remains. Therefore, even if the immobilized enzyme membrane using undenatured silk fibroin is treated with methanol for a short time, 69
Easily appear absorption peak of 698cm ^-1 in immobilized enzyme film using the regenerated silk fibroin whereas hardly appeared the absorption peak of 8 cm ^-1.

The present invention is not limited to the above-mentioned embodiments, and for example, an immunosensor, a luminescent immunosensor,
The present invention can be applied to an immobilized enzyme membrane for a multifunction sensor, various sensors for clinical tests, etc., and various design changes can be made without departing from the scope of the present invention.

[0030]

As described above, according to the invention of claim 1, since the enzyme is immobilized on the membrane containing undenatured silk fibroin as a main component in the silk gland cells derived from Lepidoptera insects, the immobilized enzyme The stability and activity retention can be improved, and when the immobilized enzyme membrane is attached to the electrode surface for measurement, the output value can be remarkably increased and the long-term storage stability can be remarkably enhanced. It has a unique effect that the peak of the output value can be obtained in a short time.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Hideo Katayama, 3 Miyukigaoka, Tsukuba, Ibaraki Daikin Industry Co., Ltd. (72) Junichiro Arai, 3 Miyukigaoka, Tsukuba, Ibaraki Daikin Industries, Ltd. ( 72) Inventor Atsushi Mizusawa 3 Miyukigaoka, Tsukuba City, Ibaraki Daikin Industry Co., Ltd.

Claims (1)

[Claims] 1. An immobilized enzyme membrane comprising an enzyme immobilized on a membrane containing undenatured silk fibroin as a main component in silk gland cells derived from Lepidoptera insects.