JP4457198B2 - Nanofibers composed of peptides and their production - Google Patents

Description

The present invention relates to nanomaterials and nanomaterial technologies composed of biomolecules composed of biological substances called peptides, and also relates to nanofabrication technologies capable of controlling the arrangement of functional substances with nanometer precision.

In recent years, research on nanometer-scale materials has progressed rapidly worldwide. In addition, environmental efforts are regarded as important, and environmentally friendly materials that model biological substances such as proteins in the natural world are being studied in order to construct devices and systems that are friendly to the global environment and humans.

However, the conventional nanoscale material is an artificial synthetic product, and it is difficult to form a regular structure with a natural product. The advantage of forming a material from a natural product is that the substance itself and its production method are close to nature and the environmental load is very small.
In addition, there are applications that make use of the characteristics of biomolecules made of biological materials, such as the ability to be used as medical materials. As a material formed by biomolecules consisting of this biological material, it is known that only a small part of proteins and peptides form fibers, but industrial conditions have not yet been established. is there.

In addition to carriers and adsorbents, nanofibers composed of peptides include, for example, medical and pharmaceutical fields as biocompatible materials, electronic / information / electronic fields as microelectronic component materials, or emulsifiers, stabilizers, and dispersants. As a wetting agent, it is expected to be used in fields such as the food industry, agriculture and forestry, and textile industry. Conventionally, spherical molecular aggregates obtained from naturally-derived phospholipids, so-called liposomes, are known as molecular aggregates of phospholipids, and this is generally the thin film method, thermal dispersion method, solution injection method, cholic acid method. Is produced by a method such as a reverse layer evaporation method, requires a skillful technique, and the obtained molecular assembly is a single membrane vesicle or a spherical multi-membrane vesicle, and a long fibrous assembly was not obtained. .

JP 2002-266007 A

  The problem to be solved by the present invention is to provide a nanoscale material or material having a regular structure made of natural products. In addition, substances such as enzymes that give various properties and functions to the nanomaterial are arranged to improve the properties and functions, and to produce materials for various applications.

  Specifically, for peptides, an array of novel peptides that form nanometer-scale fine fibers with a width of several nanometers to several tens of nanometers and a length of several hundred nanometers, and conditions for forming nanofibers of the substances that form the arrays The purpose of this invention is to provide a method for controlling the shape of nanofibers composed of peptides.

  As a result of intensive research on nanofibers composed of peptides, the present inventor has found that peptides composed of only 3 amino acids and no more than 77 amino acids in a water solvent at room temperature and normal pressure. The inventors have found that nanofibers having a width of several nanometers to several tens of nanometers and a length of several hundred nanometers to several tens of micrometers can be formed by self-assembly under specific conditions, and the present invention has been completed.

The peptide that forms the nanofiber according to the present invention is a peptide consisting of a novel amino acid sequence that has not been reported for fiber formation. Specifically, Lys-Phe-Ile-Val-Ile-Phe-Lys (SEQ ID NO: The peptide fragment or peptide fragment consisting of 3 to 7 amino acids, which is a part of the 7 amino acids shown in 1), is included. Peptide fragments include Phe-Ile-Val-Ile-Phe-Lys (SEQ ID NO: 2), Phe-Ile-Val-Ile-Phe (SEQ ID NO: 3), Ile-Val-Ile-Phe (SEQ ID NO: 4), It was confirmed that fibers could be formed with Phe-Ile-Val-Ile (SEQ ID NO: 5), Phe-Ile-Val, and Val-Ile-Phe. Further, it was confirmed that the 3 amino acid residues of Ile-Val-Ile cannot form a fiber as a peptide fragment. Based on Lys-Phe-Ile-Val-Ile-Phe-Lys (SEQ ID NO: 1), peptide fragments of 3 to 7 residues containing the natural amino acid phenylalanine (Phe) form nanofibers. I think so.

  In addition, the specific condition is to control the pH and salt strength to be specifically optimized in order to control the intermolecular force. However, Phe-Ile-Val-Ile-Phe (SEQ ID NO: 3) and Val-Ile-Phe, which are poorly soluble peptide fragments in water, are dissolved in an organic solvent and then diluted with purified water to form fibers. Was invented.

Moreover, in the manufacturing method of the fine fiber by the said peptide fragment, it discovered that the form of the fine fiber was controllable by the kind of board | substrate which produces a fine fiber, the presence or absence of washing | cleaning of a board | substrate, and the presence or absence of stirring of a liquid mixture. Moreover, it discovered that the reaction aqueous solution of supernatant could be collect | recovered by centrifuging the nanofiber comprised by the peptide which precipitates from aqueous solution system by fiber formation, and this can be reused. Almost 100% of the supernatant reaction aqueous solution can be collected and reused as it is for the production of nanofibers, so that mass production is easy and a manufacturing process that does not generate waste can be realized.

Furthermore, as a result of diligent research, enzyme immobilization materials that improve the heat resistance and active life of enzymes and allow them to function stably by binding them to nanofibers composed of peptides. It was found that the nanofiber according to the present invention can be used.
Specifically, a lysine residue of chymotrypsin, a protease (proteolytic enzyme), is biotinylated on a nanofiber consisting of 7 residues of Lys-Phe-Ile-Val-Ile-Phe-Lys (SEQ ID NO: 1). As for the substance that is bound via avidin, it has been found that proteases can suppress the state of autolysis and decrease in activity over time, and nanofiber is used as an enzyme immobilization material to stabilize the enzyme. Invented a method to use.

The peptide that forms the nanofiber according to the present invention can form nanofibers having various forms such as a linear shape and a curved shape by controlling the type and forming conditions of the amino acid sequence. By gathering the nanofibers, it is possible to form a tape-like or sheet-like larger structure on the nanometer scale.
These nanofibers, nanotapes, and nanosheets are composed only of natural amino acids and are produced using the self-assembly of peptides, so energy and artificial compounds are not required to be added. Since the solution used is completely reusable and does not produce waste in the production process, it has the effect of low environmental impact.
In addition, among the peptides forming the nanofiber according to the present invention, in particular, an enzyme is bound to a nanofiber composed of Lys-Phe-Ile-Val-Ile-Phe-Lys (SEQ ID NO: 1), which is 7 amino acid residues. Thus, it has an effect that it can be used as an immobilizing material for an enzyme having excellent heat resistance and stability.

  BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the best mode of a method for producing a peptide that forms the nanofiber of the present invention and an enzyme immobilization method will be described in detail.

  First, the procedure for producing nanofibers using peptides will be described with reference to FIG. A peptide stock solution having a concentration of 3 mmol / liter is prepared by dissolving a peptide represented by the amino acid sequence Lys-Phe-Ile-Val-Ile-Phe-Lys (SEQ ID NO: 1) in purified water. In addition, a phosphate buffer solution stock solution (around pH 7.0) and a saline solution (pH 7.0) are mixed to have a salt strength in the range of 10 to 1000 mmol / liter, preferably 500 mmol. Prepare a solution with a salt strength of 1 / liter. Using this, the peptide stock solution is diluted to 1/10 and stirred. From this, a 300 micromol / liter peptide solution (25 mmol / liter phosphate buffer, pH 7.0, containing 500 mmol / liter or less of sodium chloride) is allowed to stand at room temperature (25 degrees) for about one week. To produce nanofibers.

  A method for mass-producing the nanofiber will be described below. Since the nanofibers produced according to the above-described production procedure flow precipitate from the aqueous solution system due to fiber formation, it is possible to take out only the nanofibers from the aqueous solution by performing centrifugation or the like. It has been confirmed that the nanofibers are precipitated with a yield of nearly 100%. This means that the aqueous solution system used for the production reaction can be reused for production of the next nanofiber as it is. Therefore, mass production of the nanofiber according to the present invention can be realized by repeating the steps of introducing the target peptide into the aqueous solution system, precipitation, separation and extraction. Since the aqueous solution system accompanying the reaction is completely reusable, no waste is generated in the mass production process, and a simple mass production process can be achieved.

  FIG. 2 shows an atomic force microscope (AFM) image and FIG. 3 shows an electron microscope (EM) image of the nanofibers produced by the above-described production procedure flow (under conditions of pH 7.0, 100 mmol / liter of sodium chloride). . 4 to 6 show that the produced nanofibers have a diameter of about 5 nanometers and a length of several micrometers to several tens of micrometers.

  Next, the fact that the shape (thickness) of the nanofiber can be controlled by changing the salt strength will be described. FIG. 7 and FIG. 8 show the salt in the case of Example 1 by increasing the salt strength in the procedure for producing nanofibers from the above-mentioned peptide Lys-Phe-Ile-Val-Ile-Phe-Lys (SEQ ID NO: 1). The strength is from 100 mmol / liter to 700 mmol / liter. 7 and 8 show that the nanofiber diameter can be varied from about 5 nanometers to about 22 nanometers by adjusting the salt strength.

Similarly, as a peptide fragment, an image showing that 6 residues of Phe-Ile-Val-Ile-Phe-Lys (SEQ ID NO: 2) can form fibers is shown in FIG. 9, and Phe-Ile-Val-Ile-Phe ( FIGS. 10 to 12 show images showing that 5 residues of SEQ ID NO: 3) can form fibers, and FIGS. 10 to 12 show images showing that 4 residues of Ile-Val-Ile-Phe (SEQ ID NO: 4) can form fibers. Fig. 13 shows an image showing that 4 residues of Phe-Ile-Val-Ile (SEQ ID NO: 5) can form fibers. Fig. 14 shows an image showing that 3 residues of Phe-Ile-Val can form fibers. FIG. 15 and FIG. 17 show images showing that three residues of Val-Ile-Phe can form fibers, respectively.

Here, Phe-Ile-Val-Ile-Phe-Lys (SEQ ID NO: 2), Ile-Val-Ile-Phe (SEQ ID NO: 4), Phe-Ile-Val-Ile (SEQ ID NO: 5), Phe-Ile- Val and Val-Ile-Phe are produced by the nanofiber production procedure flow shown in FIG.

In the case of Phe-Ile-Val-Ile-Phe (SEQ ID NO: 3) or Val-Ile-Phe, which is a poorly soluble peptide, it is dissolved in a cleavage solution and diluted about 10 times with water. We have developed a method for producing nanofibers, which is prepared by allowing the prepared solution to stand at normal temperature and pressure.

In the case of Val-Ile-Phe, although it is sparingly soluble in water, it has been confirmed that nanofibers can be generated even from an aqueous solution, and it is dissolved in an aqueous solution or diluted by dissolving in an organic solvent. Alternatively, nanofibers can be produced by either production flow.

Here, as the cribage solution, any one of the following three types A to C is used.

A: 9.5 ml TFA (trifluoroacetic acid) + 0.5 ml purified water

B: 0.75 g crystalline phenol + 0.25 ml EDT (ethanedithiol) + 0.5 ml thioanisole + 0.5 ml purified water + 8.25 ml TFA

C: 0.25ml EDT + 0.25ml purified water + 9.5ml TFA

  As a next example, an example in which a peptide partially containing 7 amino acid residues whose amino acid sequence is Lys-Phe-Ile-Val-Ile-Phe-Lys (SEQ ID NO: 1) forms nanofibers will be described. To do. 19 and 20, a peptide consisting of 12 amino acids whose amino acid sequence is Ser-Ala-Gly-Lys-Phe-Ile-Val-Ile-Phe-Lys-Asn-Asp (SEQ ID NO: 6), It is the image which showed that nanofiber could be formed. For a peptide partially containing 7 amino acid residues whose amino acid sequence is Lys-Phe-Ile-Val-Ile-Phe-Lys (SEQ ID NO: 1), add an amino acid in the N-terminal direction or C-terminal direction, When the number of amino acids is increased, it has been confirmed that a nanofiber can be formed with a peptide having 77 amino acids.

  As a next example, a method for controlling the form of fine fibers by changing the type of substrate on which nanofibers are produced will be described. FIG. 11 and FIG. 12 show whether a nanofiber is formed on mica or glass when a nanofiber is produced from a peptide whose amino acid sequence is represented by Phe-Ile-Val-Ile-Phe (SEQ ID NO: 3). This shows how the shapes of the formed nanofibers are different. The one formed on mica can be formed into a sheet (a shape in which fibers of almost uniform size are arranged on one side), while the one formed on glass is a tape (fibers are bundled in a plane) Shape). This indicates that the shape of the nanofiber can be controlled depending on the type of the formation substrate.

As a next example, when the peptide is allowed to stand on the substrate at room temperature and normal pressure in the nanofiber preparation procedure, the shape of the nanofiber can be controlled by washing the substrate on which the peptide is adsorbed with water. Explain that.
FIG. 10 shows the case where the substrate was not washed when the peptide comprising the amino acid sequence Phe-Ile-Val-Ile-Phe (SEQ ID NO: 3) was left on the mica substrate. FIG. 11 shows the case where the cleaning is performed. In the case of FIG. 10, the nanofibers are formed in a mesh shape, whereas in the case of FIG. 11, the nanofibers are formed in a sheet shape with a certain direction.
This indicates that the shape of the nanofiber can be controlled under the condition whether or not the adsorbing substrate is washed with water.
Here, the substrate was washed by dropping 5 microliters of the peptide solution onto the substrate, drying it completely at room temperature, and then tilting 1 milliliter of purified water with a micropipette from the top of the substrate to about 20 microliters. It is performed by dropping liters.

As a next example, a method for controlling the form of fine fibers depending on the presence or absence of stirring of the mixed solution when producing nanofibers will be described. FIG. 17 and FIG. 18 are images showing how the shape of the formed nanofiber differs depending on the presence or absence of stirring when the nanofiber is produced from a peptide having an amino acid sequence represented by Val-Ile-Phe.

  Here, in the nanofiber image shown in FIG. 18, the number of intermolecular collisions is increased and the uniformity is increased by continuing stirring in the nanofiber generation process, and the directionality is aligned by interaction with the mica substrate ( It was shown that a nanometer-sized junction could be formed by making it possible to align with the crystal lattice direction of mica. The image in FIG. 18 shows a state in which three nanofibers having a length of about 0.5 micrometers are connected at an angular interval of 120 degrees to form a junction.

Next, by immobilizing the enzyme to the nanofiber formed by the peptide according to the present invention, the enzyme immobilization that improves the heat resistance and / or active life of the enzyme and allows the enzyme to function stably. Details of possible materials. As an example, a lysine residue or N-terminal of chymotrypsin, which is a protease (proteolytic enzyme), is used as a nanofiber consisting of 7 residues of Lys-Phe-Ile-Val-Ile-Phe-Lys (SEQ ID NO: 1). It is explained that an enzyme (protease) undergoes autolysis due to biotinylation of its amino group and bound via avidin, and the activity decreases with time. In other words, it explains that the nanofiber according to the present invention can be an enzyme immobilization material for stabilizing an enzyme. Here, a conceptual diagram of enzyme immobilization is shown in FIG.

First, with respect to the amino acid sequence Lys-Phe-Ile-Val-Ile-Phe-Lys (SEQ ID NO: 1), after formation of nanofibers, α-chymotrypsin was used in the solution as it was in a biotinylated reagent (Biotin- (AC ₅ ) ₂ -Sulfo-OSu) is used to biotinylate (part of) lysine residues. It was confirmed by measurement that the biotinylation rate per mole was about 35% for nanofibers and about 47% for chymotrypsin.

The enzyme activity is obtained by monitoring the change in absorbance of the synthetic substrate (Suc-Ala-Ala-Pro-Phe-pNA). The activity is compared under the same conditions when avidin is added to the biotinylated nanofiber and enzyme mixture (fiber and enzyme are bound) and when it is not added (fiber and enzyme are released). The measurement temperature is 25 ° C., pH 7, and it is active with a decomposition rate.
When this solution was allowed to stand at 55 ° C. for 30 minutes and then returned to 25 ° C. and the activity was measured, the activity decreased to 19.7% without avidin (about one fifth), but with avidin 49.7% ( About one-half). This indicates that the enzyme is stabilized by immobilization of the enzyme on the nanofiber, and the activity is about 2.5 times (49.7 / 19.7) at this point. FIG. 22 shows a graph showing the residual activity of α-chymotrypsin. This is data showing that nanofiber is useful as a material for producing a stable and highly functional (high heat resistant) enzyme.

Here, a method for measuring the biotinylation rate will be described in detail below. First, 4-Hydrooxyazobenzene-2-carboxylic acid (HABA) is weighed, and 10 mM NaOH aqueous solution is added so as to be 10 mM HABA (Preparation of HABA solution). Further, weigh out avidin and add phosphate buffered saline (PBS) and the previously prepared HABA solution to prepare a final concentration of avidin of about 7 μM and a final concentration of HABA of about 300 μM (HABA-avidin solution). Preparation). 180 μl of HABA-avidin solution is placed in a microcell (optical path length 1 cm), and absorbance at 500 nm is measured with an ultraviolet spectrophotometer (HITACHI U-3210 spectrophotometer). This is repeated three times, and the average of the three times is taken as that value (this is called Abs _A ). Next, 20 μl of biotinylated nanofiber is added to 180 μl of HABA-avidin solution, and the mixture is allowed to stand for 5 minutes or more, and then the absorbance at 500 nm is measured in the same manner (this is Abs _B ). From the values of Abs _A and Abs _B, the concentration of biotin immobilized on the fiber [A (mol / l)] is calculated using Equation 1 below.

If the biotinylated peptide concentration is B (mol / l), the number of biotins labeled with one peptide molecule can be calculated by the following formula 2.

Furthermore, the biotinylation rate of biotinylated chymotrypsin is also measured and calculated in the same manner.

Also, chymotrypsin can be immobilized on nanofibers by binding a biotinylated fiber and a biotinylated enzyme via avidin, respectively. Avidin has four biotin binding sites, which are used for immobilization. First, based on the concentration of biotin immobilized on the nanofibers estimated by the HABA method, an avidin solution is added in an equal amount and a binding reaction is performed for 5 minutes. Thereafter, the biotinylated chymotrypsin solution is further added so as to be equivalent to the concentration of biotin immobilized on the nanofibers to perform immobilization.

Next, a monitor measurement method for the change in absorbance of the enzyme activity will be described. In order to evaluate the substrate degradation activity of chymotrypsin immobilized on the nanofiber, the residual activity at 25 ° C. after incubation at 55 ° C. for 30 minutes is measured. Similarly, the residual activity in the absence of avidin is also measured. First, a synthetic substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF) was used as a substrate for chymotrypsin, which was added to a 25 mM phosphate buffer solution (750 mM NaCl, pH 7. 0) is dissolved to 500 μM. Further, after diluting the sample with 25 mM phosphate buffer solution (750 mM NaCl, pH 7.0) so that the concentration of chymotrypsin immobilized on the fiber becomes 3 μM, the sample prepared to 3 μM is diluted with a block incubator (KOKEN RIKA). Incubate for 30 minutes at 55 ° C. Thereafter, (A) the sample not incubated at 55 ° C. and (B) the sample incubated at 55 ° C. were simultaneously measured for activity, and the ratio (B / A) of the initial decomposition rate of the substrate AAPF was determined to determine the residual activity (% )

For the activity measurement, an ultraviolet spectroscopic plate reader (Molecular Devices) was used, and SOFT max PRO ver.3.1 (Molecular Devices) was used as measurement or analysis software. In this experiment, the absorption at 405 nm of p-nitroaniline produced from the degradation of AAPF by chymotrypsin was followed. First, 20 μl of a sample (final concentration 0.6 μM) was placed in a 96 well plate (clear flat bottom) (SUMILON), and then 80 μl of AAPF (final concentration 400 μM) was added to start the enzyme reaction. Thereafter, the 96-well plate was quickly set on a plate reader, stirred for 10 seconds, and absorbance measurement was started. The measurement time was 10 minutes, and the absorbance was measured every 10 seconds (2 seconds of stirring for each measurement). As a result, proportionality was obtained by plotting each absorbance (OD) against reaction time (sec), and the amount of change in absorbance for 5 minutes from the start of measurement was defined as the initial reaction rate (mOD / sec). . Based on each initial velocity obtained by this measurement, the residual activity was calculated.

The peptide forming the nanofiber according to the present invention can use the fiber itself as a nanomaterial or nanomaterial. Specifically, it can be used as a fiber product such as a filter or a chromatography carrier.
Moreover, since the peptide which concerns on this invention consists of a natural amino acid, it can be utilized as an environmental harmony material.
Moreover, it can utilize as a functional material, a biomaterial, and a medical material by couple | bonding with the general substance which interacts specifically regardless of other biological substances, such as protein and a nucleic acid, or organic substance and inorganic substance.

As an example of this, by modifying the peptide that forms the nanofiber according to the present invention, the peptide can be used as an enzyme immobilization material for producing a stable and highly functional (high durability) enzyme.

Flow chart of nanofiber production procedure using peptides Atomic force microscope (AFM) image of the peptide consisting of SEQ ID NO: 1 An electron microscope (EM) image of the peptide consisting of SEQ ID NO: 1. An atomic force microscope (AFM) image of the peptide consisting of SEQ ID NO: 1 showing the nanofiber diameter size (however, the formation condition is 100 mM NaCl) The graph which shows the height between horizontal direction AB in the image shown in FIG. The graph which shows the height between horizontal direction CD in the image shown in FIG. An atomic force microscope (AFM) image of the peptide composed of SEQ ID NO: 1 showing the nanofiber diameter size (however, the formation condition is 700 mM NaCl) The graph which shows the height between horizontal direction AB in the image shown in FIG. Atomic force microscope (AFM) image of the peptide consisting of SEQ ID NO: 2 Atomic force microscope (AFM) image of the peptide consisting of SEQ ID NO: 3 (on the mica substrate, without washing the substrate) Atomic force microscope (AFM) image of the peptide consisting of SEQ ID NO: 3 (on the mica substrate, with substrate cleaning) Atomic force microscope (AFM) image of the peptide consisting of SEQ ID NO: 3 (on glass substrate, with substrate cleaning) Atomic force microscope (AFM) image of the peptide consisting of SEQ ID NO: 4 Atomic force microscope (AFM) image of the peptide consisting of SEQ ID NO: 5 Atomic force microscope (AFM) image of a peptide composed of Phe-Ile-Val Atomic force microscope (AFM) image of a peptide composed of Val-Ile-Phe (solution dissolved in cleaved solution and diluted with purified water) Atomic force microscope (AFM) image of a peptide composed of Val-Ile-Phe (stationary, not stirred) Atomic force microscope (AFM) image of a peptide composed of Val-Ile-Phe (stirred) Atomic force microscope (AFM) image of a peptide composed of Ser-Ala-Gly-Lys-Phe-Ile-Val-Ile-Phe-Lys-Asn-Asp (SEQ ID NO: 6) (100 mM NaCl) Atomic force microscope (AFM) image of a peptide composed of Ser-Ala-Gly-Lys-Phe-Ile-Val-Ile-Phe-Lys-Asn-Asp (SEQ ID NO: 6) (500 mM NaCl) Conceptual diagram of enzyme immobilization Graph showing the residual activity of α-chymotrypsin

Claims (19)

  Among the peptide fragments excised from the peptide whose amino acid sequence is expressed as Lys-Phe-Ile-Val-Ile-Phe-Lys (SEQ ID NO: 1), the natural amino acid phenylalanine (Phe) is included, and the number of amino acids is 3 or more and 7 Nanofiber composed of peptides consisting of residues or less.   The nanofiber according to claim 1, wherein the nanofiber is composed of a peptide consisting of 7 residues of amino acids represented by Lys-Phe-Ile-Val-Ile-Phe-Lys (SEQ ID NO: 1).   The nanofiber according to claim 1, wherein the nanofiber is composed of a peptide consisting of 6 residues of amino acids represented by Phe-Ile-Val-Ile-Phe-Lys (SEQ ID NO: 2).   The nanofiber according to claim 1, wherein the nanofiber is composed of a peptide consisting of 5 residues of amino acids represented by Phe-Ile-Val-Ile-Phe (SEQ ID NO: 3).   The nanofiber according to claim 1, wherein the nanofiber is composed of a peptide consisting of 4 residues of amino acids represented by Ile-Val-Ile-Phe (SEQ ID NO: 4).   The nanofiber according to claim 1, wherein the nanofiber is composed of a peptide consisting of 4 residues of amino acids represented by Phe-Ile-Val-Ile (SEQ ID NO: 5).   2. The nanofiber according to claim 1, wherein the nanofiber is composed of a peptide consisting of 3 residues of amino acids represented by Phe-Ile-Val.   2. The nanofiber according to claim 1, wherein the nanofiber is composed of a peptide consisting of 3 residues of amino acids represented by Val-Ile-Phe.   It is produced by dissolving a peptide in water, mixing this with an aqueous solution having a salt strength of 10 to 1000 mmol / liter within a pH range of 2 to 10, and allowing to stand at room temperature and normal pressure. The method for producing a nanofiber according to any one of claims 2, 3, 5, 6, 7, and 8.   The method for producing nanofibers according to claim 9, wherein the aqueous solution is a mixed solution of a phosphate buffer and an alkali metal salt near neutrality (pH 7 ± 1).   9. The method according to claim 4, wherein the peptide is prepared by dissolving a peptide that is hardly soluble in water in a cleave solution, and then allowing the solution diluted with water to stand at normal temperature and pressure. Nanofiber manufacturing method.   The method for producing nanofiber according to any one of claims 9 to 11, wherein the adsorbed substrate is washed with water when allowed to stand at normal temperature and pressure.   The method for producing nanofiber according to any one of claims 9 to 11, wherein the form of the fine fiber is controlled depending on the type of substrate on which the nanofiber is produced and the presence or absence of stirring of the mixed solution.   12. The supernatant reaction aqueous solution is recovered by separating nanofibers composed of peptides that precipitate from an aqueous solution system due to fiber formation, and reused for producing nanofibers. The manufacturing method of such nanofiber.   In the process of producing nanofibers composed of peptides, by continuing to stir, the number of intermolecular collisions is increased, the uniformity is increased, and the directionality is aligned by interaction with the substrate. The nanofiber according to claim 8, wherein the nanofiber is formed.   An enzyme immobilization material characterized in that the enzyme is bound to the nanofiber according to any one of claims 1 to 8, thereby improving the heat resistance and / or active life of the enzyme and allowing the enzyme to function stably. .   9. The lysine residue or N-terminal amino group of the nanofiber according to claim 1 is biotinylated, the enzyme having a lysine residue or N-terminus is biotinylated, and the enzyme is bound to the nanofiber via avidin. An enzyme immobilization material characterized by the above.   The enzyme immobilization material according to claim 17, wherein the enzyme is chymotrypsin which is a protease (proteolytic enzyme).   The lysine residue or N-terminal amino group of the nanofiber according to claim 1 to 8 is biotinylated, and a protein or nucleic acid such as an enzyme or an antibody having a lysine residue or N-terminus is biotinylated, via avidin, An immobilizing material that is bonded to nanofibers.