Fibers with a core-shell structure immobilized with proteins

Abstract

To provide a protein-immobilized fiber, a method for producing the protein-immobilized fiber, and a reaction method using the protein-immobilized fiber, capable of achieving high protein reusability.SOLUTION: A fiber having a core-shell structure comprising a core portion and a shell portion covering the core portion is provided, in which protein is immobilized at the core portion and the shell portion contains polyamide.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to fibers having a core-shell structure with immobilized proteins, which are excellent in reusability.

Background Art

[0002] As a method of using a non-woven fabric as a carrier for immobilizing an enzyme, it is known to immobilize the enzyme on the fiber surface and use it. For example, Ran Xu et al. have immobilized laccase by covalent bonding on the fiber surface of a non-woven fabric made of polyacrylonitrile activated by acid treatment, and evaluated its function as an enzyme-immobilized carrier (Non-Patent Document 1).

[0003] In addition, reports of immobilizing an enzyme inside the fibers of a non-woven fabric and using it are also known. For example, Oeda et al. modified the side-chain carboxyl groups of polyγ-glutamic acid (PGA) with 3-glycidyloxypropyltriethoxysilane (GPTMS), which is a silica cross-linking material, and used a polymer obtained by cross-linking PGA by a sol-gel reaction of the silica part as a base material to successfully produce an enzyme-immobilized non-woven fabric in which the enzyme α-chymotrypsin was immobilized inside the non-woven fabric fibers without being denatured (Non-Patent Documents 2 and Patent Document 1). As a result, the enzyme can be immobilized inside the water-insoluble fibers without being denatured, and the immobilized α-chymotrypsin exhibited high enzyme activity. However, since a cross-linked polymer that originally gels in water is used as the base material, when considering practical use, the problem is that it has structural fragility.

[0004] Furthermore, there are reports of immobilizing enzymes within the fibers of core-shell nonwoven fabrics (Non-Patent Literature 3). In this case, a polyacrylamide polymer was used for the core component, and poly-ε-caprolactone (PCL) was used for the shell component. Although there were concerns about the permeability of substrate molecules because PCL, a hydrophobic polymer, was used for the shell component, the shell was thin (less than 200 nm thick), so the α-chymotrypsin immobilized in the core component exhibited high enzyme activity. In addition, the structural brittleness was greatly improved by using PCL as the shell. However, when considering industrial applications, it was also important that the immobilized enzyme had high reusability. When lactase was immobilized and used, the enzyme activity decreased to about 60% after 5 reuses, so improvement in this reusability was desired. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] ACS Appl. Mater. Interfaces, 5, 12554-12560(2013) [Non-Patent Document 2] Langmuir 32, 221-229 (2016) [Non-Patent Document 3] Bull. Chem. Soc. Jpn., 93, 1155-1163 (2020) [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2016-15959 [Overview of the project] [Problems that the invention aims to solve]

[0007] Enzymes are used in a variety of applications, but their stability and reusability are challenges, and technologies for immobilizing enzymes are being researched. While immobilization on the surface of a carrier is a common method, technologies for immobilizing enzymes inside fibers are also known. When immobilizing enzymes inside fibers, the structural strength of the fiber becomes an issue, and while technologies using a structurally stable material (PCL) in the shell portion of the fiber surface are known, this method has the problem of low reusability.

[0008] The object of this invention is to provide protein-immobilized fibers that can achieve high recyclability of proteins, a method for producing the protein-immobilized fibers, and a reaction method using the protein-immobilized fibers. [Means for solving the problem]

[0009] The inventors of the present invention have diligently studied to solve the above problems and have found that the above problems can be solved by using a core-shell nonwoven fabric with polyamide as the polymer material for the shell portion as an enzyme immobilization carrier. The present invention was completed based on this finding. The present invention provides the following:

[0010] <1> A fiber having a core-shell structure composed of a core portion and a shell portion covering the core portion, wherein a protein is immobilized on the core portion and the shell portion contains polyamide. <2> Proteins are enzymes. <1> The fibers described above. <3> Polyamide is nylon. <1> or <2> The fibers described above. <4> The core part contains a hydrophilic polymer. <1> from <3> The fibers listed in any one of the following. <5> The fiber diameter is 10 nm to 10 μm. <1> from <4> The fibers listed in any one of the following. <6> The thickness of the shell portion is 1 nm to 1 μm. <1> from <5> The fibers listed in any one of the following. <7> The protein content is 0.01 mg to 200 mg per gram of fiber. <1> from <6> The fibers listed in any one of the following. <8> It is a form of nonwoven fabric. <1> from <6> The fibers listed in any one of the following. <9> The method involves electrospinning using a first solution containing a hydrophilic polymer and a protein, and a second solution containing a polyamide. <1> from <8> A method for manufacturing fibers as described in any one of the following. <10> The first solution further contains a crosslinking agent. <9> Methods used. <11> <1> from <8> A reaction method comprising bringing a fiber and a substrate described in any one of the following into contact and carrying out the reaction. [Effects of the Invention]

[0011] According to the protein-immobilized fibers of the present invention, high recyclability of proteins can be achieved. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows the structure of the nanofibers that make up the core-shell nonwoven fabric (top) and PCL and nylon 6, which are the raw materials for the shell component (bottom). [Figure 2] Figure 2 shows the structures of poly(AM / DAAM) and poly(HPMA / DAMA), which are the raw materials for the core components, and ADH, which is the crosslinking agent. These react to form polyacrylamide-based crosslinked polymers poly(AM / DAAM) / ADH and poly(HPMA / DAMA) / ADH. [Figure 3] Figure 3 shows SEM images of poly(AM / DAAM) / ADH-PCL, a core-shell nonwoven fabric using PCL as the shell, and poly(AM / DAAM) / ADH-nylon 6, which uses nylon 6 as the shell (comparison of nanofiber structures of samples immediately after electrospinning (top) and after buffer immersion (bottom)). [Figure 4]Figure 4 shows a core-shell non-woven fabric using nylon 6 for the shell. TEM image of a single nanofiber of poly(AM / DAAM) / ADH-nylon 6 (measured by including a heavy metal component (sodium phosphotungstate) in the core part to obtain contrast with the shell component. The core part becomes black); measured at an acceleration voltage of 200 kV [Figure 5] Figure 5 shows the leakage behavior of fluorescein pre-encapsulated in the core nanofiber part into the immersion buffer solution. [Figure 6] Figure 6 shows the leakage behavior of FITC-modified lysozyme pre-encapsulated in the core nanofiber part into the immersion buffer solution.

Mode for Carrying Out the Invention

[0013] According to the present invention, there is provided a fiber having a core-shell structure composed of a core part and a shell part covering the core part, wherein a protein is immobilized on the core part and the shell part contains polyamide.

[0014] The fiber having a core-shell structure is a fiber having a sheath-core structure with a diameter of 1 nm to 10 μm. As the polymer material used for the core part (the part of the fiber serving as the core) and the polymer material used for the shell part (the part covering the fiber serving as the core), any different materials can be selected, but in the present invention, the shell part contains polyamide. The protein is immobilized on the core part, not on the fiber surface or the shell part.

[0015] <Protein> The protein used in the present invention may be an enzyme or a protein other than an enzyme, but is preferably an enzyme. As the enzyme, any enzyme useful as a biocatalyst can be used. Specific examples of the enzyme include, but are not limited to, hydrolase, oxidoreductase, isomerase, transferase, lyase, ligase, etc. A coenzyme may be immobilized together with the enzyme.

[0016] Examples of hydrolytic enzymes include sugar hydrolases, ester hydrolases, and peptide bond hydrolases. Examples of sugar hydrolases include glycosidase, glucosidase, amylase, lactase, pullulanase, dextranase, cellulase, hemicellulase, pectinase, xylanase, mannanase, hesperidinase, naringinase, maltase, saccharase, and lysozyme. Examples of ester hydrolases include esterase, lipase, phospholipase, nuclease, tannase, phytase, and phosphatase. Examples of peptide bond hydrolases include protease, peptidase, serine protease, cysteine ​​protease, threonine protease, aspartate protease, glutamate protease, metalloprotease, aminopeptidase, carboxypeptidase, trypsin, papain, thermolysin, chymotrypsin, and collagenase. Other examples include deaminase, glutaminase, protein glutaminase, and acylase.

[0017] Examples of oxidoreductases include alcohol dehydrogenase, glycerol dehydrogenase, glycerol phosphate dehydrogenase, lactate dehydrogenase (LDH) (also called lactate dehydrogenase), galactose dehydrogenase, glucose-6-phosphate dehydrogenase, 3-hydroxybutyrate dehydrogenase, glucose oxidase, cholesterol oxidase, galactose oxidase, choline oxidase, pyruvate oxidase, glutamate dehydrogenase, amino acid oxidase, amino oxidase, sarcosine oxidase, diaphorase, uricase, peroxidase, catalase, lipoxygenase, glucose dehydrogenase, cholesterol dehydrogenase, polyphenol oxidase, ascorbic acid oxidase, lysyl oxidase, laccase, and bilirubin oxidase.

[0018] Examples of isomerases include epimerase and racemase.

[0019] Examples of transferases include methyltransferase, carboxyltransferase, transaldolase, acyltransferase, glycosyltransferase, aminotransferase, phosphotransferase, sulfotransferase, creatine phoskinase, pirubate kinase, hexokinase, glycerol kinase, cyclodextrin glucanotransferase, and transglutaminase.

[0020] Examples of lyases include decarboxylases, aldehydeases, and dehydratases.

[0021] Examples of ligases include asparagine synthetase and glutathione synthetase.

[0022] As for the enzyme, hydrolytic enzymes are preferred, glycosidases are more preferred, and lactase can be given as an example.

[0023] Other types of proteins besides enzymes include antibodies and physiologically active peptides.

[0024] The protein content in fiber is not particularly limited, but is generally between 0.01 mg and 200 mg per gram of fiber. The lower limit of the protein content in fiber may be 0.02 mg or more, 0.05 mg or more, 0.1 mg or more, 0.2 mg or more, or 0.3 mg or more per gram of fiber. The upper limit of the protein content in fiber may be 100 mg or less, 50 mg or less, 20 mg or less, 10 mg or less, 5 mg or less, 1 mg or less, or 0.6 mg or less per gram of fiber.

[0025] <Fibers with a core-shell structure> The fiber in the present invention has a core-shell structure composed of a core portion and a shell portion that covers the core portion. As the polymer material used for the core portion, a hydrophilic polymer is preferred in order to avoid denaturing the enzyme while it is immobilized. The hydrophilic polymer may be either a non-crosslinked hydrophilic polymer or a crosslinked hydrophilic polymer.

[0026] Examples of non-crosslinked hydrophilic polymers include polyethylene glycol (polyethylene oxide), polyvinyl alcohol, metal alginate salts, methylcellulose, ethylcellulose, hydroxyethylcellulose, carboxymethylcellulose, and sodium polyacrylate. While the water solubility of these hydrophilic polymers depends on their molecular weight and the number of functional groups, it is preferable to select polymers with a molecular weight (degree of polymerization) or number of functional groups that dissolve in water as raw materials and remain insoluble in water at room temperature after molding.

[0027] Examples of crosslinkable hydrophilic polymers include hydrophilic polymers formed from polycarboxylic acids (e.g., polyγ-glutamic acid, polyα-glutamic acid, polyacrylic acid, polymethacrylic acid, carboxymethylcellulose, etc.) or polyamines (e.g., polylysine, etc.) and a crosslinking agent. One example is a crosslinked polymer obtained by reacting a hydrophilic polymer containing monomer units with ketone or aldehyde groups in their side chains, such as diacetone acrylamide (also written as DAAM) or diacetone methacrylamide (DAMA), which serve as reaction sites for the crosslinking reaction, with a dihydrazide derivative (e.g., adipic acid dihydrazide, etc.) or a diamine derivative. Hydrophilic polymers containing monomer units with ketone or aldehyde groups in their side chains may also be copolymers of monomer units with ketone or aldehyde groups in their side chains and other monomer units (e.g., acrylamide-derived units, N-(2-hydroxypropyl)methacrylamide-derived units, etc.).

[0028] The shell portion contains a polyamide. The polyamide may be either an aliphatic polyamide or an aromatic polyamide, but an aliphatic polyamide is more preferred.

[0029] Aliphatic polyamides include aliphatic nylons and their copolymers. Specifically, these include polycapramide (nylon-6), poly-ω-aminoheptanoic acid (nylon-7), poly-ω-aminononanoic acid (nylon-9), polyundecaneamide (nylon-11), polylauryl lactam (nylon-12), polyethylenediamine adipamide (nylon-2,6), polytetramethylene adipamide (nylon-4,6), polyhexamethylene adipamide (nylon-6,6), polyhexamethylene sevacamide (nylon-6,10), polyhexamethylene dodecamide (nylon-6,12), polyoctamethylene adipamide (nylon-8,6), polydecamethylene adipamide (nylon-10,8), caprolactam / lauryl lactam copolymer (nylon-6 / 12), and caprolactam. Examples include Nylon-6 / 6 / 6-ω-aminononanoic acid copolymer (Nylon-6 / 9), caprolactam / hexamethylenediammonium adipate copolymer (Nylon-6 / 6,6), lauryllactam / hexamethylenediammonium adipate copolymer (Nylon-12 / 6,6), ethylenediamineadipamide / hexamethylenediammonium adipate copolymer (Nylon-2,6 / 6,6), caprolactam / hexamethylenediammonium adipate / hexamethylenediammonium sebacate copolymer (Nylon-6 / 6,6 / 6,10), and ethyleneammonium adipate / hexamethylenediammonium adipate / hexamethylenediammonium sebacate copolymer (Nylon-6 / 6,6 / 6,10). Among the above, nylon-6 is preferred as the aliphatic polyamide.

[0030] Examples of aromatic polyamides include those obtained by polycondensation reactions of aromatic diamines such as metaxylenediamine and paraxylenediamine with dicarboxylic acids or their derivatives, such as adipic acid, suberic acid, sebacic acid, cyclohexanedicarboxylic acid, terephthalic acid, and isophthalic acid.

[0031] To allow the protein to exert its activity, it is preferable that the fiber diameter and shell thickness be small. The fiber diameter is preferably 10 nm to 10 μm. The lower limit of the fiber diameter may be 20 nm or more, 50 nm or more, 100 nm or more, 200 nm or more, 300 nm or more, or 400 nm or more. The upper limit of the fiber diameter may be 5 μm or less, 3 μm or less, 2 μm or less, 1 μm or less, 800 nm or less, or 600 nm or less.

[0032] The thickness of the shell portion is preferably 1 nm to 1 μm. The lower limit of the shell portion thickness may be 5 nm or more, 10 nm or more, 20 nm or more, 30 nm or more, or 50 nm or more. The upper limit of the shell portion thickness may be 800 nm or less, 500 nm or less, 300 nm or less, 200 nm or less, 150 nm or less, or 100 nm or less.

[0033] The fiber diameter and shell thickness mentioned above can be measured from transmission electron microscope (TEM) images of the fibers. Specifically, first, multiple fibers (for example, 30 fibers) whose cross-sections perpendicular to the length of the nanofiber can be observed in the TEM image are selected. Then, the fiber diameter and shell thickness can be measured at each location, and the average value can be calculated.

[0034] The form of the fibers of the present invention is not particularly limited and may be single fibers, fiber bundles composed of many single fibers (twisted yarn, spun yarn, braided cord, etc.), or bundled yarns formed by bundling fiber bundles. These fibers may be processed into the form of fabrics (including woven fabrics, nonwoven fabrics, etc.) woven in two or three dimensions. Preferably, the fibers of the present invention can be used in the form of a nonwoven fabric.

[0035] <Fiber manufacturing method> The fibers of the present invention can be produced by electrospinning using a first solution containing a hydrophilic polymer and a protein, and a second solution containing a polyamide.

[0036] Electrospinning allows for the easy production of fibers such as nanofibers using polymer solutions. In electrospinning, by utilizing a coaxial spinneret that enables spinning while simultaneously injecting two types of polymer solutions, it becomes possible to produce fibers with a core-shell structure, as well as core-shell nonwoven fabrics, which are aggregates of these fibers.

[0037] A first solution containing a hydrophilic polymer and a protein can be prepared by dissolving the protein in the above-mentioned solution containing the hydrophilic polymer. The concentration of the hydrophilic polymer in the first solution is not particularly limited, but is generally between 1% and 50% by mass. The concentration of the protein in the first solution is not particularly limited, but is generally between 0.01% and 50% by mass. The first solution may further contain a crosslinking agent.

[0038] As the second solution containing the polyamide, a solution obtained by dissolving the polyamide described above in a suitable solvent (for example, trifluoroethanol) can be used. The concentration of polyamide in the second solution is not particularly limited, but is generally between 1% and 30% by mass.

[0039] Electrospinning is a technique in which a polymer solution is placed in a syringe, and while a high voltage is applied between the syringe needle and collector, the polymer solution is injected. When the voltage exceeds a threshold, the repulsive force of the charges overcomes the surface tension of the polymer droplets, generating a charged jet. In the electric field, the jet extends to form extremely fine fibers, which accumulate on the collector and are spun. Electrospinning produces extremely fine fibers.

[0040] By utilizing coaxial spinnerets, electrospinning becomes possible as the polyamide in the second solution surrounds the hydrophilic polymer in the first solution, thus forming a fiber with a sheath-core structure in which the fiber surface, consisting of a protein-containing hydrophilic polymer core, is coated with polyamide as a shell layer.

[0041] <Reaction Method> The present invention provides a reaction method that includes contacting the fibers of the present invention described above with a substrate for a protein to be immobilized and carrying out a reaction.

[0042] According to the present invention, immobilized proteins can be easily recycled. That is, the fibers immobilized with the protein of the present invention can be easily recovered from the reaction mixture by filtration or the like, and after recovery, can be reused in further reactions.

[0043] The uses of the fibers of the present invention are not particularly limited, but they can be used in applications such as bioreactors that produce substances using proteins (such as enzymes) immobilized on the fibers, biosensors that detect substances using proteins (such as enzymes) immobilized on the fibers, materials for food processing, and functional fibers having functions such as deodorizing, stain-resistant, or antibacterial properties.

[0044] The present invention will be specifically described by the following examples, but the present invention is not limited to these examples. [Examples]

[0045] The following nonwoven fabrics were used for the core-shell nonwoven fabric composition. (i) A nonwoven fabric in which poly-ε-caprolactone (PCL, Figure 1, lower left) is used for the shell portion and polyacrylamide-based crosslinked polymer poly(AM / DAAM) / ADH (Figure 2) is used for the core portion containing the enzyme; (ii) A nonwoven fabric in which nylon 6 (Figure 1, bottom right) is used for the shell portion and poly(AM / DAAM) / ADH (Figure 2), a polyacrylamide-based crosslinked polymer, is used for the core portion that contains the enzyme. (iii) A nonwoven fabric in which nylon 6 (Figure 1, bottom right) is used for the shell portion and poly(HPMA / DAMA) / ADH, a polyacrylamide-based crosslinked polymer, is used for the core portion that contains the enzyme.

[0046] Example 1: Preparation of lactase-immobilized core-shell nonwoven fabric A solution was prepared as a core fiber precursor solution for nonwoven fabric by dissolving 3 mg of lactase in 2.5 mL of a 10 mM borate buffer (pH 9.2) solution of 20 wt% poly(AM / DAAM) (containing 0.5 g of poly(AM / DAAM)) and adding 124 mg of adipic acid dihydrazide (ADH). (See Figure 2 for the chemical structure of poly(AM / DAAM) / ADH.)

[0047] On the other hand, a trifluoroethanol (TFE) solution of 8% by mass of PCL or 14% by mass of nylon 6 (manufactured by Aldrich) was prepared as a shell component precursor solution.

[0048] The core fiber precursor solution and the shell component precursor solution described above were each transferred to different 10 mL syringes (Luer lock syringes). The two syringes described above were placed in different syringe pumps equipped with linear actuators (KDS-100, KD Scientific, USA) and connected via PTFE tubing to a coaxial spinneret (MECC Co. Ltd, Japan) equipped with a 27G needle (Terumo, for poly(AM / DAAM) / ADH) core solution). The above solutions were electrospun under high voltage (25 kV) at linear extrusion rates of 0.1 mL / h for poly(AM / DAAM) / ADH solution, 0.4 mL / h for PCL solution, and 0.4 mL / h for nylon 6 solution (SD-02, MECC Co. Ltd, Japan). The core component of the resulting nonwoven fabric was poly(AM / DAAM) / ADH, and the shell component was PCL or nylon 6. The distance between the tip of the coaxial spinneret and the grounded collector (aluminum plate, 150 mm × 200 mm) was 150 mm.

[0049] Example 2: Confirmation of the nanofiber structure of the nonwoven fabric <Scanning Electron Microscope (SEM) Measurement> Core-shell nonwoven fabrics were vacuum-deposited with OsO4 using plasma chemical deposition with a JEE-420T vacuum evaporator (JEOL, Japan). The fiber appearance and condensed structure of the core-shell nonwoven fabrics were evaluated by measurements using a scanning electron microscope (SEM) (JSM-6301F, JEOL, Japan). The average diameter and standard deviation of nanofibers were evaluated from SEM images of 30 nanofibers using ImageJ software.

[0050] <Transmission Electron Microscope (TEM) Measurement> The formation of the core-shell structure within the fibers was evaluated using a transmission electron microscope (TEM) with a core-shell nonwoven fabric prepared by adding sodium phosphotungstate (0.001 wt%) to the fiber precursor solution of the core portion. The addition of phosphotungstate allowed for contrast between the core and shell portions. TEM images were acquired using a JEM-z2500 instrument (JEOL, Japan) at an acceleration voltage of 200 kV.

[0051] <Result> SEM images of the obtained nonwoven fabric are shown in Figure 3, and TEN images are shown in Figure 4. From the SEM image in Figure 3, it was confirmed that, similar to the case where PCL was used as the shell component, when nylon 6 was used as the shell component, a fiber layered structure was formed within the nonwoven fabric obtained as a film, in which no fusion of fibers was observed, and the fiber diameter of each fiber was relatively uniform (496 ± 97 nm).

[0052] Furthermore, TEM images in Figure 4 show the formation of a sheath-core structure consisting of a core fiber portion and a shell portion within the fiber, and it was confirmed that the thickness of the shell portion was less than 100 nm. The fact that the nonwoven fabric fibers are so fine (less than 1 μm in diameter) and the shell portion is less than 200 nm in thickness is important for the enzyme immobilized on the core fiber portion to exhibit high activity. In addition, SEM images of this core-shell nonwoven fabric after immersion in a buffer solution (50 mM phosphate buffer (pH 7)) for 3 days are shown in Figure 3, and no fusion of fibers or significant changes in the fiber system were observed, suggesting that the core-shell structure was maintained.

[0053] Compared to the case using PCL, using nylon 6 tended to result in a lower rate of fiber fusion during nonwoven fabric fabrication (each nanofiber remained separate). Furthermore, TEM measurements (Figure 4) revealed the shell and core nanofiber portions, confirming that the fabric had a double-layered structure with the shell portion covering the core fibers, as expected.

[0054] Example 3: Evaluation of enzyme retention capacity and permeability of low molecular weight substrates of core-shell nonwoven fabric. A characteristic of core-shell nonwoven fabric-type enzyme immobilization carriers is that (large molecular weight) enzyme molecules can be retained without leakage from the core nanofiber portion, while small molecular weight substrate molecules can penetrate the shell portion and reach the core nanofiber portion where the enzyme molecules are trapped. We evaluated whether this property is also exhibited in nonwoven fabrics using nylon 6 as the shell. Here, we substituted lysozyme fluorescently labeled with FITC for the enzyme molecule and fluorescein for the low molecular weight substrate, and prepared core-shell nonwoven fabrics in which each was pre-encapsulated in the core nanofiber portion. We then evaluated the leakage behavior when these fabrics were immersed in a buffer solution.

[0055] Figure 5 shows that for low molecular weight molecules (fluorescein, MW 332), efficient leakage was observed immediately after immersion in the buffer solution. This result suggests that for low molecular weight substrates, rapid exchange with the material dissolved in the immersion buffer is possible regardless of whether PCL or nylon 6 is used as the shell. On the other hand, for high molecular weight proteins (lysozyme, MW 1.4 kDa), Figure 6 shows that almost no leakage into the external solution was observed.

[0056] Example 4: Evaluation of the enzymatic activity of immobilized lactase <Evaluation of lactase enzyme activity> Nonwoven fabrics immobilized with lactase of different weights were prepared (the immobilized amount was 0.39 mg (enzyme) / g (nonwoven fabric) for those using PCL as the shell, and 0.46 mg (enzyme) / g (nonwoven fabric) for those using nylon 6 as the shell). These were immersed in 50 mM phosphate buffer (pH 7) at room temperature for 150 minutes and shaken (150 rpm). This removed any lactase that had not been immobilized within the fibers. Subsequently, the remaining nonwoven fabrics were used to perform a hydrolysis reaction with o-Nitrophenyl-β-D-galactopyranoside as the substrate. Nonwoven fabrics of different weights and 4 mL of 50 mM phosphate buffer (pH 7) were added to 10 mL sample vials, and then o-Nitrophenyl-β-D-galactopyranoside was added to a final concentration of 6 mM. After reacting at 37°C for 20 minutes (stirring the solution with a stirrer tip), the nonwoven fabric was removed from the solution, and the reaction was stopped by adding 1 mL of 500 mM sodium carbonate aqueous solution. The amount of o-Nitrophenol produced was quantified by absorption spectroscopy, and the enzyme activity was evaluated by plotting the amount of enzyme contained (mg) on ​​the x-axis and the amount of o-Nitrophenol produced per minute (nmol) on the y-axis using linear approximation, and the slope (nmol / (min mg)). The removed nonwoven fabric was washed with 50 mM Phosphate buffer (pH 7) and reused in the same experiment. This reuse experiment was performed four times. Between the second and third reuses, the enzyme-immobilized nonwoven fabric was washed and dried and left at 4°C for 3 days before use. A comparison was also made with the activity when the same amount of lactase was dissolved in the buffer solution. These results are summarized in Tables 1 and 2.

[0057] [ka]

[0058] [Table 1]

[0059] [Table 2]

[0060] Table 1 shows that in the case of lactase-immobilized nonwoven fabric using nylon 6 as the shell, the activity (approximately 100%) was almost the same as when the same amount of enzyme was dissolved in the solution during the first use, and even after four further reuses, the activity was maintained at over 80% (80.3%). On the other hand, Table 2 shows that in the case of lactase-immobilized nonwoven fabric using PCL as the shell, the activity was slightly less than half (48.3%) of when the same amount of enzyme was dissolved in the solution during the first use, and after four further reuses, the activity decreased to about 30% (30.0%).

[0061] On the other hand, regarding changes in enzyme activity when reused, when nylon 6 was used as the shell, there was a tendency for the decrease in activity during reuse to be smaller compared to when PCL was used as the shell.

[0062] Example 5: Lactase-immobilized core-shell nonwoven fabric (Manufacturing method) A solution was prepared as a core fiber precursor solution for nonwoven fabric by dissolving 6 mg of lactase in 2.5 mL of a 100 mM phosphate buffer (pH 8.0) solution of 20 wt% poly(AM / DAAM) (containing 0.5 g of poly(AM / DAAM)) and adding 187 mg of adipic acid dihydrazide (ADH). (See Figure 2 for the chemical structures of poly(AM / DAAM) and ADH.)

[0063] On the other hand, a trifluoroethanol (TFE) solution of 8% by mass of PCL or 10% by mass of nylon 6 (manufactured by Aldrich) was prepared as a shell component precursor solution.

[0064] The core fiber precursor solution and the shell component precursor solution described above were each transferred to different 10 mL syringes (Luer lock syringes). The two syringes described above were placed in different syringe pumps equipped with linear actuators (KDS-100, KD Scientific, USA) and connected via PTFE tubing to a coaxial spinneret (MECC Co. Ltd, Japan) equipped with a 27G needle (Terumo, for poly(AM / DAAM) / ADH) core solution). The above solutions were electrospun under high voltage (25 kV) at linear extrusion rates of 0.2 mL / h for poly(AM / DAAM) / ADH solution, 0.8 mL / h for PCL solution, and 1.0 mL / h for nylon 6 solution (SD-02, MECC Co. Ltd, Japan). The core component of the resulting nonwoven fabric was poly(AM / DAAM) / ADH, and the shell component was PCL or nylon 6. The distance between the tip of the coaxial spinneret and the grounded collector (aluminum plate, 150 mm × 200 mm) was 150 mm.

[0065] (evaluation) Similar to the method described in Example 4, <Evaluation of Lactase Enzyme Activity>, ten sets of reuse experiments were performed (10 repeated experiments). As a result, the lactase-immobilized nonwoven fabric with PCL as the shell showed a relative enzyme activity (%) of 80% after 10 reuses. Furthermore, the lactase-immobilized nonwoven fabric with Nylon 6 as the shell showed a relative enzyme activity (%) of 90% after 10 reuses. In Example 5, the use of phosphate buffer pH 8.0 as the solvent accelerated the crosslinking reaction by ADH, which is thought to have prevented enzyme leakage outside the fibers and maintained high enzyme activity.

[0066] Example 6: Lipase-immobilized core-shell nonwoven fabric (Manufacturing method) A solution was prepared as a core fiber precursor solution for nonwoven fabric by dissolving 25 mg of FITC-modified lipase in 2.5 mL of a 100 mM phosphate buffer (pH 8.0) solution of 20 wt% poly(AM / DAAM) (containing 0.5 g of poly(AM / DAAM)) and adding 187 mg of adipic acid dihydrazide (ADH). (See Figure 2 for the chemical structures of poly(AM / DAAM) and ADH.)

[0067] On the other hand, a trifluoroethanol (TFE) solution of 8% by mass of PCL or 10% by mass of nylon 6 (manufactured by Aldrich) was prepared as a shell component precursor solution.

[0068] The core fiber precursor solution and the shell component precursor solution described above were each transferred to different 10 mL syringes (Luer lock syringes). The two syringes described above were placed in different syringe pumps equipped with linear actuators (KDS-100, KD Scientific, USA) and connected via PTFE tubing to a coaxial spinneret (MECC Co. Ltd, Japan) equipped with a 27G needle (Terumo, for poly(AM / DAAM) / ADH) core solution). The above solutions were electrospun under high voltage (25 kV) at linear extrusion rates of 0.2 mL / h for poly(AM / DAAM) / ADH solution, 0.8 mL / h for PCL solution, and 1.0 mL / h for nylon 6 solution (SD-02, MECC Co. Ltd, Japan). The core component of the resulting nonwoven fabric was poly(AM / DAAM) / ADH, and the shell component was PCL or nylon 6. The distance between the tip of the coaxial spinneret and the grounded collector (aluminum plate, 150 mm × 200 mm) was 150 mm.

[0069] (Evaluation 1) Shape retention in water Nonwoven fabric cut to approximately 5 mm x 5 mm was placed in a 2.0 mL Eppendorf tube and immersed in approximately 1.5 mL of deionized water. After immersion at room temperature for 7 days, the appearance and fiber diameter were confirmed using SEM imaging. As a result, the lipase-immobilized nonwoven fabric with PCL as its shell dissolved, but the lipase-immobilized nonwoven fabric with Nylon as its shell maintained its shape. The fiber diameter was 422 ± 115 nm before immersion in deionized water and 475 ± 98 nm after immersion in deionized water.

[0070] (Evaluation 2) Hydrolysis activity evaluation A predetermined amount of lipase-immobilized nonwoven fabric was immersed in 2 mL of 50 mM phosphate buffer (pH 7). 60 μL of acetone solution of 60 mM p-nitrophenylbutyric acid was added, and the reaction was carried out at 37°C for 30 minutes. The amount of lipase-immobilized nonwoven fabric added was determined based on the amount of lipase contained within, resulting in 5, 10, 15, 20, and 25 μg of lipase being added to each reaction solution. After the reaction, the nonwoven fabric was separated from the reaction solution, and the amount of reaction product under each reaction condition was evaluated from the absorbance derived from p-nitrophenolate produced as the hydrolysis reaction progressed. Enzyme activity was evaluated by defining 1 unit (nmol / (mg min)) as the activity of 1 mg of enzyme hydrolyzing substrate molecules (nmol) per minute. The same experiment was repeated three times. The results are summarized in Table 3.

[0071] [Table 3]

[0072] As shown in Table 3, hydrolytic activity of lipase was confirmed when using either PCL or Nylon 6. Regarding hydrolytic activity, Nylon 6 showed higher activity and a higher retention rate of activity after repeated use.

[0073] (Evaluation 3) Evaluation of transesterification activity 0.5 mmol of (±)-1-phenylethanol and 2.5 mmol of vinyl acetate were dissolved in 2.5 mL of tert-butyl methyl ketone, and a predetermined amount of lipase-immobilized nonwoven fabric was immersed in this solution. After 48 and 100 hours at 37°C, the reacted nonwoven fabric was separated from the reaction solution, and the reaction rate was calculated using 1H-NMR, and the asymmetric selectivity of the reaction was evaluated using a chiral column (Daicel Corporation, CHIRALCELL OD-H). One unit (μmol / (mg hour)) was defined as the amount of substrate molecules (μmol) that 1 mg of enzyme transesterifies per hour. The results are shown in Table 4.

[0074] [Table 4]

[0075] As shown in Table 4, transesterification activity was confirmed regardless of whether PCL or Nylon6 was used. Furthermore, asymmetric selectivity was maintained.

[0076] Example 7: Lactate dehydrogenase (LDH) immobilized core-shell (Poly(HPMA / DAMA)) nonwoven fabric (Manufacturing method) A solution was prepared as a core fiber precursor solution for nonwoven fabric by dissolving 5 mg of FITC-modified LDH in 2.0 mL of 100 mM phosphate buffer (pH 7.0) containing 25 wt% poly(HPMA / DAAM) (HPAA:DAMA=8:2) (Poly(HPMA / DAMA) is non-cytotoxic), and adding 57.6 mg of adipic acid dihydrazide (ADH). (See Figure 2 for the chemical structures of poly(HPMA / DAMA) and ADH). HPMA stands for N-(2-hydroxypropyl)methacrylamide. DAMA stands for diacetone methacrylamide.

[0077] On the other hand, a 10% by mass solution of nylon 6 (manufactured by Aldrich) in trifluoroethanol (TFE) was prepared as a shell component precursor solution.

[0078] The core fiber precursor solution and the shell component precursor solution described above were each transferred to different 10 mL syringes (Luer lock syringes). The two syringes described above were placed in different syringe pumps equipped with linear actuators (KDS-100, KD Scientific, USA) and connected via PTFE tubing to a coaxial spinneret (MECC Co. Ltd, Japan) equipped with a 27G needle (Terumo, for poly(HPMA / DAAM) / ADH) core solution. The above solutions were electrospun under high voltage (25kV) at linear extrusion rates of 0.3 mL / h for the poly(HPMA / DAAM) / ADH solution and 1.2 mL / h for the nylon 6 solution (SD-02, MECC Co. Ltd, Japan). The core component of the resulting nonwoven fabric was poly(HPMA / DAAM) / ADH, and the shell component was nylon 6. The distance between the tip of the coaxial spinneret and the grounded collector (aluminum plate, 150 mm × 200 mm) was 150 mm.

[0079] (Evaluation of activity) A predetermined amount of LDH-immobilized nonwoven fabric (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg) was immersed in 1 mL of 0.2 M Tris-HCl acid buffer (pH 8) while shaking for 3 hours. Then, the reaction solution (320 mM L-lactic acid, 150 mM 1-MPMS, 6.4 mM NAD) was added. + 1 mL of 2.6 mM WST-1 was added, and the mixture was reacted at room temperature for 30 minutes under light protection. After stopping the reaction by adding 1 mL of 1 M acetic acid, the absorbance at 438 nm was measured.

[0080] The measured enzyme activity was 1.01 U [nmol / (mg min)]. However, the mass used here is the mass of the nonwoven fabric, not the mass of the enzyme. Lactate dehydrogenase activity was confirmed in a nonwoven fabric containing lactate dehydrogenase using poly(HPMA / DAMA), which is non-cytotoxic, as the core material.

Claims

1. A fiber having a core-shell structure composed of a core portion and a shell portion covering the core portion, wherein a protein is immobilized on the core portion and the shell portion contains polyamide. Proteins are enzymes, and they are fibers.

2. The fiber according to claim 1, wherein the polyamide is nylon-6.

3. The fiber according to claim 1 or 2, wherein the fiber diameter is 10 nm to 10 μm.

4. The fiber according to claim 1 or 2, wherein the thickness of the shell portion is 1 nm to 1 μm.

5. The fiber according to claim 1 or 2, wherein the protein content is 0.01 mg to 200 mg per gram of fiber.

6. The fiber according to claim 1 or 2, in the form of a nonwoven fabric.

7. A method for producing fibers according to claim 1 or 2, comprising electrospinning using a first solution containing a hydrophilic polymer and a protein, and a second solution containing a polyamide.

8. The method according to claim 7, wherein the first solution further comprises a crosslinking agent.

9. A reaction method comprising bringing a fiber and a substrate according to claim 1 or 2 into contact and carrying out a reaction.

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