JP3044239B2 - Immobilized bioactive substance using keratin protein as carrier and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immobilized bioactive substance on which a bioactive substance such as an enzyme is immobilized, which can be used for a biosensor, a bioreactor or the like. More specifically, the present invention relates to an immobilized bioactive substance in which a keratin protein thin film is formed on the surface of a fiber constituting a fiber assembly, and the thin film contains a bioactive substance.

[0002]

2. Description of the Related Art Biosensors are attracting attention as important technologies supporting biotechnology in the 21st century. An enzyme that specifically acts on a specific chemical substance is immobilized on a polymer gel film, which is coated on the surface of an underlying electrode such as an oxygen electrode or hydrogen peroxide electrode, and a solution containing a substrate is spotted. Then, the low-molecular-weight substrate diffuses into the enzyme-immobilized carrier, chemically reacts with the enzyme contained in the enzyme-immobilized carrier, and changes in the output current value corresponding to the amount of chemical reaction from the electrode. This is the principle of biosensor construction. Biosensors are widely used in the fields of fermented food industry, chemical analysis in clinical tests, industrial processes, environmental measurement, food, medicine, pharmacy and the like.

[0003] As a carrier for immobilizing an enzyme which is a biocatalyst, a biopolymer protein can be used. After dissolving insect-derived biopolymer silk fibroin fiber in a concentrated aqueous neutral salt solution such as calcium chloride, calcium carbonate, lithium bromide, and lithium thiocyanate, dialyzing inorganic substances and ions such as calcium, chlorine, and lithium Japanese Patent Application Laid-Open No. 61-18 / 1988 attempts to physically entrap and immobilize an enzyme using a regenerated silk fibroin obtained by removal by means of a carrier.
It is disclosed in JP-A-7790. A gel layer of silk fibroin is coated on the constituent surface of a hydrophilic fiber aggregate, and an immobilized physiologically active substance in which the gel layer contains a physiologically active substance and a method for producing the same are disclosed (JP-A-2-24518).
No. 9). According to the present invention, the regenerated silk fibroin is used as a carrier for immobilization, and the hydrophilic fiber aggregate is immersed in an aqueous solution of silk fibroin containing a physiologically active substance, taken out and dried, and then the immobilized carrier is insolubilized. In order to perform the treatment, it was necessary to go through a step of immersing the fiber aggregate coated with the insolubilized silk fibroin in, for example, 80% alcohol for 30 seconds. Further, the fiber material used in the publication is limited to hydrophilic fibers, and the hydrophobic fiber material is not a target of the fiber assembly. As described above, since the material of the fiber assembly is limited to hydrophilic, and it is necessary to repeat three steps of immersion treatment, insolubilization treatment, and drying treatment, the process for preparing the immobilized physiologically active substance is complicated. Further, there has been a problem that the activity of the immobilized fluorescent substance is reduced during the insolubilization treatment using an organic solvent. An immobilized enzyme using keratin as an immobilized carrier is disclosed in JP-A-52-57391. This immobilized enzyme is obtained by adding an enzyme (acid phosphatase) to a keratin aqueous solution prepared by dissolving and dialyzing feathers with sodium thioglycolate, and then forming a film on an acrylic plate. Since this film is water-soluble, the keratin film is immersed in an aqueous formaldehyde solution to form intermolecular cross-links, thereby making it insoluble in water.
In this method, since the film is formed on a flat plate, the surface area of the film is limited. Therefore, a solution containing a substrate such as a test solution and the enzyme are not sufficiently contacted with each other, and the rate of the enzyme reaction cannot be increased. In addition, there is also a problem that the enzyme activity is reduced by cross-linking water insolubilization treatment with formaldehyde or the like after film formation (see Comparative Example 5).

[0004]

An object of the present invention is to provide an immobilized bioactive substance having a high activity. Another object of the present invention is to provide a method for producing the immobilized physiologically active substance by a simple method.

[0005]

SUMMARY OF THE INVENTION The present invention provides a biosensor for biosensors as a part of the present inventors' elucidation of the chemical structure, dissolution characteristics, and physicochemical characteristics of keratin proteins while developing new uses in the non-clothing field. Selection of a carrier for immobilization,
We have conducted intensive studies on the method of immobilizing a physiologically active substance, the method of dissolving wool, the detection accuracy of a test substance, and the like, and an aqueous solution of keratein obtained by dissolving keratin fibers represented by wool, S-
By using carboxymethylkeratein aqueous solution, keratose aqueous solution, etc. as a raw material of a carrier for immobilizing a physiologically active substance, an immobilized physiologically active substance excellent in comprehensiveness of the physiologically active substance and detection characteristics of a test substance in a low concentration region can be obtained. They found that they could be prepared and completed the present invention.

That is, the present invention is characterized in that a keratin protein thin film is formed on the surface of the constituent fibers of the fiber assembly, and the thin film contains a physiologically active substance.
The gist is an immobilized physiologically active substance using keratin protein as a carrier.

In the present invention, physiologically active substances include enzymes,
Including bacteria, organelles, cells and tissues. The fiber aggregate that can be used in the present invention is a fiber aggregate made of organic or inorganic, synthetic or natural fibers. It may be a fiber aggregate laid on a flat surface with continuous fibers,
It may be a fiber aggregate in the form of a woven fabric, a knitted fabric, a nonwoven fabric, or the like. The material may be a synthetic material such as nylon, tetron or acrylonitrile, or a cellulosic natural material, or a protein fiber such as wool or silk fiber. Alternatively, a synthetic fiber or a natural fiber aggregate may be used, and examples of the material include polypropylene, polyethylene terephthalate, polyamide, and a polyolefin material. Further, it may be a fiber aggregate composed of a plurality of mixed materials such as polyamide / polyolefin, polyester / nylon and the like. Particularly excellent materials constituting the fiber assembly include cellulose-based materials, which are hydrophilic natural materials. A high-performance industrial wiper (Sanbacto (trade name), manufactured by Asahi Kasei Corporation) containing 35% rayon and other polyacrylonitrile can be exemplified. Further, a fiber aggregate of ultra-fine fibers containing a polyester material as a main component, Toraysee Miracle Cloth (trade name, manufactured by Toray Industries, Inc.) can be used in the same manner as Sanbact.
Alternatively, a feature is that a fiber material of an inorganic substance such as fine glass fiber can be used. In order to improve the reactivity between the substrate and the physiologically active substance, a fiber aggregate as fine as possible and having a large effective surface area is desirable.

In the immobilized physiologically active substance of the present invention, a thin film of keratin protein is formed on the surface of the fiber assembly, and the physiologically active substance is immobilized in the thin film. Here, keratin protein includes keratin treated with a reducing agent to cut its disulfide bond -SS- to form a thiol group -SH; keratin; an organic compound having a hydrogen atom of the thiol group of keratein having a functional group. Keratin derivatives substituted with a group; and keratose treated with an oxidizing agent to break disulfide bonds and to make water soluble. Examples of the organic group having a functional group include an alkyl group such as a methyl group, an ethyl group, and a propyl group, a benzyl group, an aralkyl group such as a phenylethyl group, a carboxymethyl group, a carboxylethyl group, and a substituted alkyl group such as an aminoethyl group. Groups are exemplified. Among these groups, an organic group having a hydrophilic functional group is preferable, and a carboxymethyl group is particularly preferable. Examples of the raw material of the keratin protein used for inclusion include animal hair fibers such as wool, alpaca, cashmere, mohair, and angora. Wool is preferred, and if it is wool, it may be of the Japan Cordel variety or of the Australian Merino, Bolworth and Corrider species. Keratin proteins such as horns, hooves, hair, feathers and the like can be similarly used. Keratin proteins contain a large number of hydrophilic amino acid residues rich in polar groups such as basic amino acids and acidic amino acids. Proteins composed of highly polar amino acids are excellent in water absorption and water retention, and have good affinity with biocatalysts and living cells. Therefore, when the keratin protein of the present invention is used as a carrier for immobilizing an enzyme, there is a feature that the comprehensiveness of a physiologically active substance is improved. Amino acid analysis of keratin protein revealed that it contained 5.4 mol% cystine (Cys),
Contains 10 mol% or more of basic amino acid residues such as lysine (Lys), arginine (Arg) and histidine (His), and contains 18 mol% of acidic amino acids such as aspartic acid (Asp) and glutamic acid (Glu). On the other hand, in the silkworm silk fibroin, the total content of Cys is 0, Lys, Arg, His, etc. is 0.76 mol%, and the total content of Asp, Glu is 2.49 mol%.
Which is significantly different from the amino acid composition of the keratin protein.

Next, a method for producing the immobilized physiologically active substance of the present invention will be described. First, a method for preparing an aqueous solution of keratin will be described. As described above, keratin protein contains 5.4 mol% of Cys, but silk fibroin does not. Cys, an amino acid residue, forms a cross-link between keratin molecules. Intermolecular cross-linking by Cys is a feature not found in silk fibroin molecules. Since animal hair fibers such as wool have intermolecular cross-links due to cystine, keratin fibers do not dissolve in a neutral salt solution unlike silk fibroin. In order to dissolve keratin fibers, it is necessary to first cut Cys bonds between molecules using a reducing agent such as mercaptoethanol or thioglycolic acid under a nitrogen atmosphere to reduce the keratin molecules to make them solubilized. When mercaptoethanol is used, the reduction treatment is preferably performed in a urea solution. The urea concentration is 7.
The optimum is 5 to 8.8M, preferably 7.8 to 8M.
When thioglycolic acid is used, 1 to 4% of NaCl
Should be added. For example, when using mercaptoethanol as a reducing agent, keratin fibers are immersed in a urea aqueous solution having the above concentration, and after degassing, under a nitrogen atmosphere, 45 ° C or less, desirably 20 to 25 ° C, mercaptoethanol into 10 g wool. Add 3-5 ml, and stir for 2-3 hours. Thereafter, the pH is adjusted to about 10.5 and the mixture is further stirred for about 3 hours. In this way, the keratin molecules are reduced, and keratein in which the -SS- bond between the molecules has been cleaved to form SH is obtained. Dialysis using pure water to remove urea and excess mercaptoethanol yields an aqueous solution of keratein.

The keratein having an SH group thus reduced can be further reacted with an alkylating agent, for example, a (substituted) alkyl halide to be used as an S- (substituted) alkylkeratein. Alkylation may be performed according to a known method. As an example, a case where iodoacetic acid is used as an alkylating agent will be described. The above reduced keratein is added at a temperature of 20 to 25 ° C. in a nitrogen atmosphere,
While stirring, 10 to 17 g of iodoacetic acid (molecular weight: 185.95) is added to 10 g of wool and reacted. 1 to
After 2 hours, the pH is adjusted to 8.5 and the excess iodoacetic acid is removed by dialysis against pure water to obtain an aqueous solution of S-carboxymethylkeratein.

Keratose obtained by treating keratin fibers with an oxidizing agent can also be used. This reaction is performed as follows. First, 9.5 ml of formic acid (HCOOH) is 30%
After mixing with 0.5 ml of an aqueous hydrogen peroxide solution (commercially available, H ₂ O ₂ ), the mixture was allowed to stand at 25 ° C. for 2 hours, cooled to −10 ° C., and used as a liquid A. Next, 5 ml of formic acid and 1 ml of methanol are mixed and cooled to −10 ° C., which is used as a liquid B. The solution A and the solution B are mixed and maintained at -10 ° C, and about 200 mg of wool is treated in this mixed solution for 4 hours to completely dissolve the wool. The resulting solution is dialyzed against pure water using a cellulose dialysis membrane, diluted with water at 0 ° C. to 350 ml, and freeze-dried to prepare a water-soluble and powdery keratose. An aqueous solution of keratose can be easily prepared by dissolving a certain amount of powdered keratose in distilled water. (Hereinafter, the aqueous solution of keratein, the aqueous solution of S-alkylated keratein and the aqueous solution of keratose may be simply referred to as an aqueous solution of keratin or an aqueous solution of keratin protein). The concentration of the keratin aqueous solution is desirably a dilute solution of 3% or less as measured by a dry weight method. Most preferably 0.
It is a 3-0.8% keratin protein aqueous solution.

The keratin protein aqueous solution is added at a concentration of 1% by weight.
４０40%, preferably 5-20% of a physiologically active substance is added, dissolved at 25 ° C. with gentle stirring, and left standing for about 2 hours,
A keratin aqueous solution containing a physiologically active substance is obtained.

A keratin protein is used as a carrier for immobilization, for example, cholesterol oxidase as a physiologically active substance,
In order to construct an efficient and accurate biosensor for measuring cholesterol by immobilizing cholesterol esterase, water-insoluble cholesterol generated when the test solution reacts is dispersed well in water. That is the decisive factor. For this purpose, if a surfactant is added to a keratin protein aqueous solution containing a physiologically active substance, the output can be improved, and the dispersion of measured values in a measurement experiment can be reduced. Examples of the surfactant include Triton X-100 (trade name, Wako Pure Chemical), Tween-80, and Cremophor (trade name, Aldrich). The addition amount of the surfactant is 0.00 in weight concentration.
The concentration is preferably 1 to 0.1%, particularly preferably 0.001%.
~ 0.01%.

Next, the aqueous keratin solution containing the above-mentioned physiologically active substance is applied to the fiber assembly by an appropriate method such as impregnation, application, and dropping. For example, 0.8 μl thereof is dropped on a fiber assembly of a predetermined size placed on a polyethylene membrane by using a safety pipetter for a trace amount, and left at room temperature for 10 minutes. next,
The fiber assembly is heated to 30 to 60 ° C, particularly preferably 45 to 50 ° C.
Light drying at 60 ° C for 60 minutes is recommended. In order to reduce the variation of the measurement data and immobilize as many physiologically active substances as possible on the fiber aggregate, instead of dropping the aqueous solution of keratein with a high concentration of the physiologically active substance onto the fiber aggregate at once,
As described above, it is desirable to repeat the method of repeatedly dropping the physiologically active substance solution on the fiber assembly that has been lightly dried.
The inclusive state of the physiologically active substance can be changed by the concentration of the physiologically active substance, the concentration of the aqueous keratin solution, the amount of the aqueous keratin solution dropped onto the fiber assembly, and the like. Can be controlled. Thus, a thin film containing a physiologically active substance in the keratetin carrier is formed on the surface of the fiber assembly. In the present invention, the activity of the immobilized physiologically active substance does not substantially decrease because it is not necessary to perform any water insolubilization treatment on the thin film by a chemical reaction using a reagent such as an aldehyde.

The immobilized physiologically active substance of the present invention can be suitably used for biosensors and bioreactors. For example, glucose oxidase, cholesterol oxidase,
When a physiologically active substance such as uricase is immobilized, and the immobilized physiologically active substance of the present invention is coated on the base surface of the hydrogen peroxide electrode, the concentration of glucose, cholesterol, uric acid and the like can be measured.

[0016]

The immobilized physiologically active substance of the present invention has a keratin protein having good hydrophilicity and excellent incorporation of the physiologically active substance coated on the surface of the constituent fibers of the fiber aggregate as an immobilizing carrier, and is subjected to insolubilization treatment. The gel layer is formed in the state of the undenatured keratin protein that has not been subjected. Therefore, a large membrane area can be obtained as compared with a planar membrane, and the same can be used whether the constituent fiber material of the fiber assembly is hydrophobic or hydrophilic. For example, when a substrate acts, the measured values are less scattered, and an immobilized physiological substance having high output responsiveness and high activity can be produced. The immobilized bioactive substance according to the present invention has a bioactive substance immobilized on a carrier containing keratin protein as a main component, and the bioactive substance is physically immobilized on the carrier. Since no addition was made, the decrease in activity was slight. Further, it is possible to provide an immobilized enzyme membrane having performance capable of detecting even a low concentration of a substance to be measured.

[0017]

The present invention will be described in more detail with reference to the following examples. Examples 1-3 The pigments and fats contained in Merino wool (64'S) were removed by a 2.5 hour soxhlet extractor treatment using a mixed solution of benzene-ethanol 50/50 / vol%. did. A rubber tube from a dry nitrogen cylinder was connected to one mouth of the three-neck flask via a three-way cock. Reaction system
To adjust the pH, the pH electrode is always inserted into another port, and the other port is used for charging a necessary drug. Fiber length is about 1
8.18 g of wool fiber chopped to a size of 1 cm was put into a 1-liter three-necked flask, and 450 ml of an 8M urea solution was added thereto. The operation of purging with nitrogen gas, reducing the pressure in the three-necked flask to about 45 mmHg with an aspirator for 15 minutes, and rapidly returning to the atmospheric pressure was repeated three to four times. In this way, the air contained between the keratin fibers in the three-necked flask can be completely removed, and the reaction between the aqueous urea solution and the keratin molecules becomes efficient. After the replacement with nitrogen was completed, 4.8 ml of mercaptoethanol was added as a reducing agent into the three-necked flask, and the mixture was allowed to stand in an 8M aqueous urea solution for 2 to 3 hours. Further, the pH of the mixed solution in the three-necked flask was adjusted to 10.5 by adding a small amount of about 100 ml of a 5N KOH solution. Allowed for 3 hours at room temperature to allow the wool fibers to completely dissolve. A solution of fibrous wool fibers is an aqueous solution of keratein. The aqueous solution of keratein was dialyzed against pure water for 2 days using a cellulose dialysis membrane. Three types of aqueous keratein solutions having different concentrations were prepared while drying with air or by adding pure water.

The different concentrations (0.
Glucose oxidase (hereinafter referred to as GOD) is added to each of the 07%, 0.1% and 0.7% aqueous keratein solutions.
(Manufactured by Toyobo Co., Ltd.) was dissolved at a concentration of 10% to obtain an aqueous solution of keratein containing GOD. This aqueous solution
8 μl of Toraysee (trade name, 5 × 3 mm) placed on a polyethylene membrane with a safety pipetter for trace amount
The mixture was dropped on a fiber assembly manufactured by Toray Industries, Inc. and left at room temperature for 10 minutes. Next, the fiber assembly was dried at 50 ° C. for 60 minutes to dry it lightly to obtain immobilized glucose oxidase.

Examples 4 to 6 A 0.01% aqueous solution of keratein 45 obtained in Example 1
To 0 ml, 9.5 g of iodoacetic acid was added at room temperature, and the S-carboxymethylation reaction of keratein was performed for 1 hour. 5
The pH of the keratin solution was adjusted to 8.5 with an aqueous NKOH solution. During the reaction, the three-necked flask was shielded from light with an aluminum foil so as not to be stimulated by light or the like. Thus, an aqueous solution of S-carboxymethylkeratein was obtained. The S-carboxymethylkeratein aqueous solution was dialyzed against pure water for 2 days using a cellulose permeable membrane. Three types of aqueous solutions of S-carboxymethyl keratein having different concentrations (0.07%, 0.1%,
(0.7%). Thereafter, in the same manner as in Example 1, an immobilized glucose oxidase having glucose oxidase immobilized in a keratin film on the fiber assembly was obtained.

Comparative Examples 1-2 A solution containing 10% GOD (Comparative Example 1) without using the aqueous solution of keratein and the aqueous solution of S-carboxymethylkeratein (Comparative Example 1), or 0.8% of 0.01% surfactant (Triton X) 100, trade name, Wako Pure Chemical) and 10% GOD
An immobilized glucose oxidase was prepared by impregnating 0.8 μl of an aqueous solution containing the compound (Comparative Example 2) into Toraysee in the same manner as in Example 1.

Comparative Examples 3 and 4 A silkworm cocoon thread was mixed with an ethanol / benzene mixed solution using a Soxhlet extractor to remove wax and pigments contained in the sample. The sample was replaced with 0.2% of Marcel soap and 0.05 of sodium carbonate. % Sericin in the outer layer of the cocoon thread was removed by treating the mixed solution at 98 ° C. for 30 minutes. After being completely dried at 60 ° C., it was treated with 8 M lithium bromide at 55 ° C. for 30 minutes. Dissolve the solution using a cellulose dialysis membrane.
It was dialyzed against pure water at 4 ° C. for 4 days. By blowing and drying, a regenerated silk fibroin solution having a concentration of 0.07% and 0.4% was prepared. In the same manner as in Example 1, a silk fibroin solution containing 10% GOD was impregnated in Toraysee and dried to prepare immobilized glucose oxidase.

Examples 7 to 12 Merino wool (64'S) used in Example 1 from which pigments and fats had been removed was solubilized with thioglycolic acid as follows. The nitrogen connection and the pH electrode connection to the three-necked flask were the same as described in Example 1. 3.2g shredded
Was added to a 500 ml three-necked flask, into which 150 ml of a 3% NaCl solution was placed. The operation of purging with nitrogen gas, reducing the pressure in the three-necked flask to about 45 mmHg with an aspirator for 15 minutes, and rapidly returning to the atmospheric pressure was repeated three to four times. Nitrogen gas replacement was performed for 20 minutes. 0.2 M thioglycolic acid was added to the system for dissolving wool, the pH was adjusted to 10 by further adding aqueous ammonia, and the mixture was reacted for 3 hours in a nitrogen-substituted environment using a stirrer to dissolve the wool. An aqueous keratein solution was prepared by filtering under nitrogen gas to remove unreacted wool. The aqueous solution of keratein was put into a cellulose dialysis membrane and dialyzed for 2 days at 15 ° C. It was blow-dried and concentrated to a predetermined concentration to obtain an aqueous solution of keratein.

Separately from this, an aqueous ammonia solution is added to the aqueous wool keratein solution before being blown and dried.
The pH was adjusted to 8, and 0.2M iodoacetic acid was further added under nitrogen gas, and the mixture was stirred with a stirrer for 15 minutes. In this way, S-carboxymethylation of keratein enables S-carboxymethylation.
An aqueous carboxymethylkeratein solution was prepared. The aqueous solution of S-carboxymethylkeratein was dialyzed against pure water for 2 days in a 15 ° C. environment to remove unreacted substances using a cellulose dialysis membrane. It was blow-dried and concentrated to a predetermined concentration to obtain an experimental aqueous solution of S-carboxymethylkeratein. Prepared in this way and adjusted to three types of aqueous solutions of keratein having different concentrations by blast drying or aqueous solutions of S-carboxymethylkeratein (0.07%, 0.4% and 0.7%, respectively). Example 1
10% of glucose oxidase (GOD, manufactured by Toyobo) was dissolved in the same manner as in the above to obtain an aqueous solution of keratein or an aqueous solution of S-carboxymethylkeratein containing 10% of GOD. 0.8 μl of this aqueous solution was added dropwise to Toraysee using a microsafe pipettor, and dried at 50 ° C. for 60 minutes to prepare immobilized glucose oxidase.

Comparative Example 5 In the same manner as in Example 3, 0.8 μl of a 0.7% aqueous keratein solution prepared so as to have a GOD concentration of 10% was added dropwise to Toraysee in the same manner as in Example 3, and this was added at room temperature. For 1 hour and further dried for 1 hour in a constant temperature dryer at 40 ° C. The Tracy on which the thin film of the water-soluble keratein thus prepared was formed was added to a 0.5% aqueous solution of kultaraldehyde in water.
After immersion at 0 ° C. for 15 minutes, keratein containing immobilized glucose oxidase was subjected to water insolubilization treatment by further drying at room temperature for 3 hours.

Performance Evaluation (1) Evaluation Using Glucose Aqueous Solution The immobilized glucose oxidase obtained in Examples 1 to 6 and Comparative Examples 1 to 5 was mounted on the surface of a hydrogen peroxide electrode, and a predetermined concentration of As the output characteristics when 0.8 μl of the glucose aqueous solution was dropped, the output current (μA) from the hydrogen peroxide electrode, the time when the output current was maximum (hereinafter referred to as peak time), and the coefficient of variation (CV%) were determined. Was. The number of measurements was eight, and the results are averaged and shown in Table 1. The coefficient of variation was determined by the following equation. CV (%) = δ _n-1 / X × 100 where n is the number of data (8), δ _n-1 is n−1 standard deviations, and X is the average value of the measured data. The CV value is an index indicating how the data varies (a larger CV value indicates a greater variation state).
The degree of penetration of the glucose solution when a glucose test solution of a predetermined concentration was spotted was determined by visual measurement as follows. The results of the determination are also shown in Table 1. The symbols shown in Table 1 mean the following. Permeability of aqueous glucose solution into immobilized glucose oxidase (evaluated in 4 steps): + A little aqueous glucose solution penetrates. ++ Glucose aqueous solution permeates normally. +++ Aqueous glucose solution penetrates well. ++++ The aqueous glucose solution penetrates very well.

[0026]

[Table 1]

The results in Table 1 can be summarized as follows. When water-soluble S-carboxymethylkeratein is used as the immobilized enzyme carrier, a higher output current value is obtained than when water-insoluble S-carboxymethylkeratein or conventionally used silk fibroin is used. It exhibits excellent responsiveness as a biosensor because of small variations in measured values and a short peak time. It is confirmed that the keratin protein of the present invention as an immobilized physiologically active substance carrier exhibits an excellent effect. The permeability (hydrophilicity) of the immobilized glucooxidase using S-carboxymethylkeratein as a carrier was extremely good. If the glucose concentration is 1
When increasing from 00 mg / dl to 300 and 600 mg / dl, the output current value from the hydrogen peroxide electrode increased almost linearly as seen from the following equation, and a proportional relationship was recognized between the two. This means that when quantifying glucose as a substrate with immobilized glucose oxidase using the keratin protein according to the present invention as a carrier, even if the substrate is at a low concentration, an output current proportional to the glucose concentration can be accurately detected. doing. Glucose concentration (mg / dl; X) solution and output current value (μA;
Correlation with Y): 0.07% aqueous carrier solution: Y = 0.45111X + 0.0415R = 0.99002 0.4% aqueous carrier solution: Y = 0.66333X + 0.01225 R = 0.99
416 0.7% aqueous carrier solution: Y = 0.73056X + 0.040994 R = 0.99
022 In the above equation, R indicates a correlation coefficient. The immobilized glucose oxidase of Example 3, which was not subjected to the water insolubilization treatment,
The output value is excellent, the variation state is small, and the peak time is short as compared with that of Comparative Example 5 which has been subjected to the water insolubilization treatment. This shows that the solidified carrier is not subjected to the water insolubilization treatment, whereby glucose in the test solution is easily diffused and reacts favorably with the immobilized physiologically active substance.
Although not shown in the table, the immobilized enzymes obtained in Examples 7 to 12 also obtained excellent output characteristics similar to those in Table 1.
The permeation state of the test liquid was also good.

(2) Evaluation Using Serum The immobilized glucooxidase obtained in Examples 1 to 6 and Comparative Examples 1 and 2 was mounted on the surface of a hydrogen peroxide electrode, and a liquid control serum (Wako Pure Chemical Industries, Ltd.) was placed thereon. , Glucose concentration 80
mg / dl), the output current (μA) from the peroxide electrode when 0.8 μl of serum prepared so as to contain a predetermined concentration of glucose was dropped, and the time during which the output current becomes maximum (hereinafter, peak time) ) And the coefficient of variation (CV%). Also, the degree of penetration of serum penetrating into the Toraysee fiber when the serum was dropped was evaluated. The number of measurements was eight, and the results obtained are shown in Table 2 on average. The indication in Table 2 showing the permeability was evaluated in four steps as in (1).

[0029]

[Table 2]

When serum was added dropwise to a physiologically active substance immobilized on a conventional carrier such as silk fibroin, the peak value of the current generally fluctuated and it was difficult to obtain a regular waveform, but keratin protein was used as the carrier. With the immobilized physiologically active substance of the present invention, variations in output current could be reduced, and a clear waveform with less noise could be obtained. In addition, according to the above-described conventional method, generally, even when a serum having a low glucose concentration was dropped, an output waveform could not be measured, but in the present invention, even in a serum having a glucose concentration of about 100 mg / dl, the glucose in the serum was not sufficient. Measurement was possible, and peaks appeared in all the measurements. Although not shown in the table, Example 7
As for the immobilized enzymes obtained in Nos. To 12, excellent output characteristics similar to those in Table 2 were obtained, and the permeation state of the test liquid was also good.

[0031]

Since the present invention has the above-described structure, it exhibits the following effects. 1) Unlike conventional methods that use silk fibroin, which is originally a biopolymer, as a carrier for immobilizing a bioactive substance, there is no need to insolubilize the immobilized bioactive substance. There is no risk of deactivation. 2) The immobilized physiologically active substance of the present invention has high hydrophilicity,
Therefore, the wet state with respect to the substrate solution, serum, plasma and whole blood is remarkably better than that of the conventional one, and the permeation state is also good. 3) Since a keratin film is formed on the surface of the constituent fibers of the fiber-based composite having irregularities, a sufficiently large net film area can be secured. Therefore, contact with the test solution containing the substrate of the physiologically active substance is sufficiently performed, and a high reaction rate can be obtained. 4) Detectable even at low concentrations of physiologically active substances, eg, substrates. 5) Even if the constituent surface of the fiber aggregate is hydrophobic or hydrophilic, the same can be used. Therefore, a wide fiber aggregate can be used.

Continuation of front page (56) References JP-A-52-57391 (JP, A) JP-A-2-245189 (JP, A) JP-A-5-308969 (JP, A) JP-A-60-120988 (JP) JP-A-63-160585 (JP, A) JP-A-1-240189 (JP, A) (58) Fields studied (Int. Cl. ⁷ , DB name) C12N 11/04

Claims (6)

(57) [Claims] A keratin protein thin film is formed on the surface of a fiber assembly, and a physiologically active substance is included in the thin film. material. 2. The immobilized bioactive substance according to claim 1, wherein the keratin protein is keratein or keratose. 3. The immobilized physiologically active substance according to claim 2, wherein the keratein is a keratein derivative in which at least a part of the H atom of the SH bond is substituted with an organic group having a functional group. 4. The immobilized bioactive substance according to claim 3, wherein the organic group is a carboxymethyl group, an aminoethyl group or a phenylethyl group. 5. An immobilization method comprising treating a surface of a fiber assembly with an aqueous solution of keratein or a derivative thereof or a keratose containing a physiologically active substance to form a thin film of keratein or a derivative thereof or keratose on the surface. A method for producing a chemical physiologically active substance. 6. The method according to claim 5, wherein the treatment comprises the steps of dripping, coating or impregnating, and drying.