Enzyme-encapsulating complex and method for producing same

Abstract

To provide complexes including an enzyme in which the enzyme included in a fibrous resin can efficiently exert its catalytic activity.SOLUTION: A complex including an enzyme comprises: a first resin member including a plurality of laminated fibrous resin sheets and having a recess formed in the center; an enzyme complex including a porous body and an enzyme supported on the porous body and accommodated in the recess; and a second resin member including a plurality of laminated fibrous resin sheets and covering the enzyme complex. The first resin member and the second resin member are joined together at their peripheral portions. The meshes of the first resin member and the second resin member are smaller than the enzyme complex.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present application relates to an enzyme-encapsulating complex in which an enzyme is encapsulated in a fibrous resin, and a method for producing the same. [Background technology]

[0002] A method for producing an enzyme-encapsulated fibrous resin by deforming and fixing the surface of an enzyme-supported fibrous resin so that the surfaces interlock is known (Patent Document 1). However, when an enzyme is encapsulated in a general fibrous resin by the method described in Patent Document 1, there is a risk that the enzyme will fall out of the fibrous resin because many types of enzymes are smaller than the mesh of the surrounding fibrous resin. To address this issue, it is conceivable to make the mesh of the fibrous resin extremely small so that the enzyme will not fall out of the fibrous resin. However, in a fibrous resin with a mesh that prevents the enzyme from falling out, it becomes difficult for the liquid to act on the encapsulated enzyme to pass through the fibrous resin, and the efficiency of the enzyme treatment decreases. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2018-99885 A Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present application is to provide an enzyme-encapsulated complex in which an enzyme encapsulated in a fibrous resin is unlikely to leak out of the fibrous resin and is able to exert its catalytic activity. [Means for solving the problem]

[0005] The enzyme-encapsulating complex of the present application comprises a plurality of stacked fibrous resin sheets, a first resin member having a recess formed in the center, a porous body, and an enzyme supported on the porous body, an enzyme complex contained in the recess, and a plurality of stacked fibrous resin sheets, a second resin member covering the enzyme complex, the first resin member and the second resin member being joined at their peripheries, and the mesh of the first resin member and the second resin member being smaller than that of the enzyme complex.

[0006] A method for producing an enzyme-encapsulated complex in one embodiment of the present application includes an enzyme complex placement step of placing an enzyme complex larger than the mesh of a first resin member having a plurality of stacked fibrous resin sheets, the first resin member having a central recess, a porous body, and an enzyme supported on the porous body, in the recess; and an enzyme complex encapsulation step of covering the enzyme complex with a second resin member having a plurality of stacked fibrous resin sheets and a mesh smaller than the enzyme complex, bonding the peripheries of the first resin member and the second resin member to encapsulate the enzyme complex between the first resin member and the second resin member to obtain an enzyme-encapsulated complex.

[0007] A method for producing an enzyme-encapsulated complex in another aspect of the present application includes a porous body arrangement step of arranging a porous body larger than the mesh of a first resin member, which comprises a plurality of stacked fibrous resin sheets and has a recess formed in the center, in a recess of the first resin member; a porous body encapsulation step of covering the porous body with a second resin member which comprises a plurality of stacked fibrous resin sheets and has a mesh smaller than the porous body, and joining the peripheries of the first resin member and the second resin member to encapsulate the porous body between the first resin member and the second resin member; and an enzyme support step of contacting a liquid containing an enzyme with the porous body after the porous body encapsulation step, to support the enzyme in the porous body and obtain an enzyme-encapsulated complex. Effect of the Invention

[0008] In the enzyme-encapsulated complex of the present application, an enzyme complex comprising a porous body and an enzyme is encapsulated in a fibrous resin sheet having a mesh smaller than that of the enzyme complex. Therefore, the enzyme can be immobilized in the fluid, and the fluid passes near the enzyme, facilitating interaction between the enzyme and the reaction substrate in the fluid. Furthermore, the mechanical strength of the enzyme is high. In addition, according to the method for producing the enzyme-encapsulated complex of the present application, the enzyme-encapsulated complex of the present application can be easily produced. [Brief description of the drawings]

[0009] [Figure 1] FIG. 2 is a conceptual diagram showing a method for producing an enzyme-encapsulating complex according to an embodiment. [Diagram 2] SEM image of PET nonwoven fabric. [Diagram 3] SEM images of the bottom surface of the first resin member in each example [Figure 4] (a) SEM image of SBA-15-type mesoporous silica with a pore diameter of 4 nm, (b) SEM image of SBA-15-type mesoporous silica with a pore diameter of 8 nm. [Diagram 5] 1 is an image of the disk cartridge (enzyme-encapsulating complex) of Example 1. [Figure 6] 2 is an evaluation device for the disk cartridges of the respective examples and comparative examples. [Figure 7] 10 is a graph showing the relationship between the amount of reaction substrate solution delivered using the disk cartridge of Example 1 and the decolorization rate of the reaction substrate solution recovered in the recovery container. [Figure 8] 13 is a graph showing the relationship between the amount of reaction substrate solution delivered using the disc cartridge of the comparative example and the decolorization rate of the reaction substrate solution recovered in the recovery container. [Figure 9] 13 is a graph showing the relationship between the amount of reaction substrate solution delivered using the disk cartridge of Example 2 and the decolorization rate of the reaction substrate solution recovered in the recovery container. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The enzyme-encapsulating complex of the embodiment of the present application includes a first resin member, a second resin member, and an enzyme complex. The first resin member and the second resin member include a plurality of laminated fibrous resin sheets. The fibrous resin sheet is a thin plate-like material formed by assembling resin fibers, and includes a mesh that is a gap surrounded by the fibers. From the viewpoint of easy plasticization of the resin by impregnation with carbon dioxide, the resin constituting the fibrous resin sheet is preferably a thermoplastic resin, particularly an amorphous thermoplastic resin or a thermoplastic resin with a reduced degree of crystallization. Examples of the amorphous thermoplastic resin or the thermoplastic resin with a reduced degree of crystallization include ABS resin, acrylic resin, polyethylene terephthalate (PET), polycarbonate (PC), polyvinyl chloride (PVC), polystyrene (PS), cyclic olefin copolymer, cycloolefin polymer, and polypropylene (PP).

[0011] A recess is formed in the center of the first resin member. That is, in the first resin member, a recess is formed in the center of one side of the laminated fibrous resin sheets. The "center" of the first resin member and the second resin member does not necessarily have to be the center, but refers to a part other than the periphery. The recess in the center of the first resin member can be formed, for example, by overlapping a laminate of multiple fibrous resin sheets with a hole formed in the center with a normal laminate of multiple fibrous resin sheets with fibrous resin also present in the center, and bonding them together.

[0012] The enzyme complex comprises a porous body and an enzyme. The enzyme is supported by the porous body. Examples of the enzyme include oxidoreductase, hydrolase, transferase, lyase, isomerase, and synthase. In order to facilitate fixation of the enzyme in the porous body, the porous body is preferably one having a function of immobilizing the enzyme or protein in the pores. An example of such a porous body is mesoporous silica. In addition, since the enzyme is easily supported, the pore diameter of the porous body is preferably 1 nm to 50 nm. Note that this pore diameter is a number average value measured by a nitrogen gas adsorption method.

[0013] In order to prevent the enzyme complex from coming out of the first resin member and the second resin member made from a general fibrous resin sheet, the particle size of the porous body is preferably 0.1 μm to 200 μm. The particle size of the porous body is a number average value measured by dynamic light scattering and SEM observation. The enzyme complex is accommodated in a recess in the first resin member. The second resin member covers the enzyme complex. In order to firmly fix the enzyme complex to the first resin member, a convex portion corresponding to the recess in the first resin member may be formed in the center of the second resin member. "Corresponding" means that the convex portion fits into at least a part of the recess. By providing the second resin member with this convex portion, the enzyme complex can be fixed to the recess in the first resin member.

[0014] In the enzyme-encapsulated complex of this embodiment, the first resin member and the second resin member are bonded at the periphery. Therefore, it is possible to prevent the enzyme complex from protruding from between the first resin member and the second resin member. If it is possible to prevent the enzyme complex from protruding, it is not necessary for the periphery of the first resin member and the second resin member to be bonded all around. In the enzyme-encapsulated complex of this embodiment, the mesh of the first resin member and the second resin member is smaller than that of the enzyme complex.

[0015] The enzyme-encapsulating complex usually encapsulates a plurality of enzyme complexes. The mesh of the first resin member and the second resin member does not need to be smaller than all of the enzyme complexes encapsulated in the enzyme-encapsulating complex. Depending on the application of the enzyme-encapsulating complex, as long as the number of enzyme complexes fixed by the first resin member and the second resin member is sufficient, the mesh of the first resin member and the second resin member may be larger than some of the enzyme complexes.

[0016] The mesh size of the first and second resin members being smaller than that of the enzyme-encapsulated complex or the porous body refers to a size relationship between the mesh size of the first and second resin members and the enzyme-encapsulated complex or the porous body such that when the enzyme-encapsulated complex or the porous body is placed on the first and second resin members in a floating state without applying an external force, for example, without stretching or compressing, the enzyme-encapsulated complex or the porous body does not pass through the first and second resin members. A method for producing such a first and second resin member and a method for joining the first and second resin members will be described later.

[0017] FIG. 1 conceptually illustrates a method for producing an enzyme-encapsulated complex according to a first embodiment of the present application. The method for producing an enzyme-encapsulated complex according to the first embodiment includes a first resin member producing step, a second resin member producing step, an enzyme complex arranging step, and an enzyme complex encapsulating step. The first resin member producing step includes a first plasticizing step, a first deformation step, and a first fixing step. In the first plasticizing step, a plurality of (e.g., 20) fibrous resin sheets 10 are stacked and placed in a lower mold 12, and an upper mold 14 is lowered to approach the plurality of fibrous resin sheets 10. In this state, carbon dioxide in a liquid state or a high-density gaseous state is introduced between the lower mold 12 and the upper mold 14 via a gas flow path 16, and the carbon dioxide is brought into contact with the plurality of fibrous resin sheets 10 to plasticize the surfaces of the plurality of fibrous resins 10. The high-density gaseous state is a gaseous state that will liquefy if the volume is reduced even slightly.

[0018] In the first deformation step, the upper mold 14 is further lowered to deform the plurality of fibrous resins 10 with their surfaces plasticized by pressure. In the first fixing step, in a state in which the plurality of fibrous resins 10 are deformed, carbon dioxide is removed from between the lower mold 12 and the upper mold 14 via the gas flow path 16 to fix the plurality of fibrous resins 10 to obtain a first resin member 18. By appropriately adjusting the material, quantity, and fiber diameter of the fibrous resins 10, the pressing force of the upper mold 14 against the lower mold 12, and the flow rate of carbon dioxide, etc., a first resin member 18 having a mesh size smaller than that of the enzyme-encapsulated complex or the porous body can be obtained. The same applies to the size of the mesh of the second resin member 28 described later.

[0019] The first resin member 18 includes a plurality of stacked fibrous resin sheets 10, and has a recess 18a formed in the center. In the first plasticization step of the first embodiment, in order to form the recess 18a in the center of the first resin member 18, a laminate of a plurality of fibrous resin sheets 10b having a hole 10a formed in the center and a laminate of a plurality of fibrous resin sheets 10c having fibrous resin also present in the center are stacked and brought into contact with carbon dioxide in a liquid state or a high-density gaseous state.

[0020] The second resin member manufacturing process includes a second plasticization process, a second deformation process, and a second fixing process. In the second plasticization process, a plurality of fibrous resin sheets 20 are stacked and placed in a lower mold 22, and an upper mold 24 is lowered to approach the plurality of fibrous resin sheets 20. In this state, carbon dioxide in a liquid state or a high-density gaseous state is introduced between the lower mold 22 and the upper mold 24 via a gas flow path 26 to bring the carbon dioxide into contact with the plurality of fibrous resin sheets 20, thereby plasticizing the surfaces of the plurality of fibrous resin sheets 20.

[0021] In the second deformation process, the upper die 24 is further lowered to deform the plurality of fibrous resins 20 with their surfaces plasticized by pressing. In the second fixing process, in a state in which the plurality of fibrous resins 20 are deformed, carbon dioxide is removed from between the lower die 22 and the upper die 24 via the gas flow path 26 to fix the plurality of fibrous resins 20 and obtain a second resin member 28. In the second deformation process of this embodiment, the plurality of fibrous resins 20 are pressed and deformed by the upper die 24, which has a recess 24a formed in its center, so that a convex portion 28a is formed in its center.

[0022] In the enzyme complex placement step, the enzyme complex 30 is placed in the recess 18a of the first resin member 18. The enzyme complex 30 includes a porous body and an enzyme supported by the porous body. As described above, the enzyme complex 30 is larger than the mesh of the first resin member 18. In the enzyme complex encapsulation step, the enzyme complex 30 is covered with the second resin member 28, and the peripheries of the first resin member 18 and the second resin member 28 are joined to encapsulate the enzyme complex 30 between the first resin member 18 and the second resin member 28 to obtain the enzyme encapsulation complex 40. As described above, the second resin member 28 includes meshes smaller than the enzyme complex 30.

[0023] More specifically, the enzyme complex encapsulation step is performed as follows. First, the second resin member 28 is placed on the first resin member 18 on which the enzyme complex 30 is placed so that the protrusions 28a are fitted to the recesses 18a. Next, in this state in which the first resin member 18 and the second resin member 28 are overlapped, the first resin member 18, the second resin member 28, and the enzyme complex 30 are accommodated in the lower mold 32. Then, in a state in which the upper mold 34 is lowered and brought close to the second resin member 28, carbon dioxide in a liquid state or a high-density gaseous state is introduced between the lower mold 32 and the upper mold 34 via the gas flow path 36, so that the carbon dioxide comes into contact with the first resin member 18 and the second resin member 28, and their surfaces are plasticized.

[0024] Next, the upper mold 34 is further lowered, and the first resin member 18 and the second resin member 28 whose surfaces have been plasticized are deformed by pressure. Then, with the first resin member 18 and the second resin member 28 in a deformed state, carbon dioxide is removed from between the lower mold 32 and the upper mold 34 via the gas flow path 36, and the plurality of fibrous resins 20 are fixed. In this way, the enzyme can be fixed even in the fluid, and the fluid passes near the enzyme, facilitating interaction between the enzyme and the reaction substrate in the fluid, thereby obtaining an enzyme-inclusion complex 40 in which the mechanical strength of the enzyme is high.

[0025] According to the method for producing an enzyme-inclusion complex of the first embodiment, a high-temperature environment that may deactivate the enzyme is not required. According to the method for producing an enzyme-inclusion complex of the first embodiment, the enzyme-inclusion complex can be produced using carbon dioxide, which is highly safe and approved as a food additive. Therefore, the enzyme-inclusion complex can be applied to enzyme-substrate reactions in the food or pharmaceutical fields. The enzyme-inclusion complex 40 can also be produced by the following production method in which the enzyme is later supported on the porous body.

[0026] That is, the method for producing an enzyme-encapsulated complex according to the second embodiment of the present application includes a porous body arrangement step, a porous body encapsulation step, and an enzyme support step following the porous body encapsulation step. In the porous body arrangement step, a porous body larger than the mesh of the first resin member 18 is arranged in the recess 18a of the first resin member 18. In the porous body encapsulation step, the porous body is covered with a second resin member 28 having a mesh smaller than that of the porous body, and the peripheries of the first resin member 18 and the second resin member 28 are joined together to encapsulate the porous body between the first resin member 18 and the second resin member 28.

[0027] In the enzyme carrying step, a liquid containing an enzyme is brought into contact with the porous body to carry the enzyme in the porous body, thereby obtaining an enzyme-inclusion complex 40. In the process of bringing the liquid containing an enzyme into contact with the porous body, for example, the first resin member 18 and the second resin member 28 in which the porous body is encapsulated are immersed in the liquid containing the enzyme, or the liquid containing the enzyme is passed through the first resin member 18 and the second resin member 28 in which the porous body is encapsulated. EXAMPLES

[0028] We created a disk cartridge that is an enzyme-encapsulated complex that encapsulates mesoporous silica carrying azoreductase (AzoR). Using this disk cartridge, we performed the reduction and decomposition reaction of an azo dye (methyl red) shown in the chemical reaction formula below, and evaluated the activity of the enzyme in the disk cartridge.

[0029] [ka]

[0030] Example 1 (Disc cartridge manufacturing) The second resin member was prepared by the following procedure. Polyethylene terephthalate resin (TK3, Bell Polyester Products) was applied to a sheet having a basis weight of 30 g / m 2(Nippon Nozzle Co., Ltd.) to obtain a PET nonwoven fabric with an average fiber diameter of 4 μm. This PET nonwoven fabric was then laser processed to obtain a circular sheet with a diameter of 31 mm. An SEM image of this circular sheet is shown in Figure 2. As shown in Figure 2, this circular sheet has pores (mesh) with a size of 10 μm or more, which is larger than the particle size of general mesoporous silica, which is 0.1 μm to 10 μm.

[0031] Twenty of these circular sheets were stacked and placed in a cylindrical cup-shaped metal lower mold with an inner diameter of 32 mm and a depth of 50 mm. A piston, which is a metal upper mold with a recess of 18 mm in diameter and 0.6 mm in depth formed on the underside, was brought close to the stack of circular sheets, and carbon dioxide in a gaseous state at a temperature of 25°C was filled between the lower mold and the piston at cylinder pressure (vapor pressure). The piston was further lowered, and the stack of circular sheets was pressed so that the peripheral part had a thickness of 0.7 mm. Carbon dioxide was removed while pressing, and a second resin member with a convex part in the center was obtained.

[0032] The first resin member was also produced by the following procedure. A circular sheet, which was the same as the raw material of the second resin member, was laser processed to obtain an annular sheet with a circular hole of 18 mm in diameter formed in the center. This stack of 20 annular sheets was placed on top of a stack of 20 circular sheets, which were the same as the raw material of the second resin member. These 40 sheets were placed in the lower mold used in the production of the second resin member. A piston, which is a metal upper mold with a convex portion of 18 mm in diameter and 1.5 mm in thickness formed on the lower surface, was brought close to the stack of annular sheets, and carbon dioxide in a gaseous state at a temperature of 25°C was filled between the lower mold and the piston at cylinder pressure (vapor pressure). The piston was further lowered to press the stack of annular sheets and circular sheets so that the peripheral portion was 2.2 mm thick. Carbon dioxide was removed while pressing to obtain a first resin member with a recess in the center.

[0033] FIG. 3 shows an SEM image of the bottom surface of the first resin member. In addition, FIG. 4(a) and FIG. 4(b) show SEM images of SBA-15 type mesoporous silica (Sigma-Aldrich) with pore sizes of 4 nm and 8 nm, respectively. As shown in FIG. 3, FIG. 4(a), and FIG. 4(b), the fibrous resin constituting the first resin member becomes dense by pressing the fibrous resin sheet, and the mesh of the first resin member is smaller than that of general mesoporous silica. Therefore, this mesoporous silica and this mesoporous silica carrying enzymes are less likely to flow out of the first resin member. The size relationship between the mesh of the first resin member and general mesoporous silica is also the same for the second resin member.

[0034] Next, the enzyme complex was prepared by the following procedure. The AzoR gene amplified from the chromosomal DNA of Escherichia coli was inserted into the pET100 / D-TOPO vector to prepare a circular plasmid DNA for AzoR expression. This circular plasmid DNA was introduced into recombinant E. coli, and the E. coli protein expression system was used to express AzoR in Escherichia coli. We produced AzoR derived from coli (dimer, number of amino acid residues: 201, molecular weight (monomer): approximately 23 kD).

[0035] 100 mg of the above-mentioned SBA-15 type mesoporous silica with a pore size of 4 nm was weighed into a 5 mL tube. A 0.25 mg / mL AzoR solution was prepared using the above-mentioned AzoR and 25 mM Tris-HCl buffer (pH 7.5). The above-mentioned mesoporous silica and AzoR solution were gently mixed using a rotator at a temperature of 4°C for 17 hours. Furthermore, the mixture was centrifuged at a temperature of 4°C and 12,000 rpm for 10 minutes, and the supernatant was removed.

[0036] 4 mL of 25 mM Tris-HCl buffer (pH 7.5) was added to this, and the precursor of AzoR-supported mesoporous silica was resuspended and washed. After the above centrifugation and supernatant removal were performed again, the mixture was suction filtered using filter paper. The mixture was dried at 30°C for 1 hour in a thermostatic bath to obtain AzoR-supported mesoporous silica (pore diameter 4 nm), which is an enzyme complex. AzoR-supported mesoporous silica (pore diameter 8 nm) was also obtained in the same manner using SBA-15 type mesoporous silica with a pore diameter of 8 nm.

[0037] Then, a disk cartridge was produced by the following procedure. 10 to 50 mg of the enzyme complex was placed in the recess of the first resin member, and the second resin member was placed on top of it, and then placed in the lower mold used in producing the second resin member. A piston, which is a metal upper mold with a flat lower surface, was brought close to the second resin member, and gaseous carbon dioxide at a temperature of 25°C was filled between the lower mold and the piston at cylinder pressure (vapor pressure). The piston was further lowered, and the first resin member and the second resin member were pressed so that the overall thickness was 1.8 mm. Carbon dioxide was removed while pressing, and a disk cartridge was obtained. An image of the disk cartridge held by tweezers is shown in Figure 5.

[0038] (evaluation) The evaluation device used to verify the catalytic activity of the azoreductase in the disk cartridge is shown in Figure 6. In Figure 6, a part of the image is enlarged and its cross section is shown as a schematic diagram. Using this evaluation device, it was verified whether the azo dye that passed through the disk cartridge prepared above was reductively decomposed. In other words, it was evaluated whether a disk cartridge in which the enzyme functions could be prepared by decolorizing the azo dye.

[0039] The disk cartridge was set in the sample holder container in an incubator kept at 30°C. A reaction substrate solution containing 0.05 mM methyl red, 25 mM Tris-HCl buffer (pH 7.5), 0.3 mM NADH (nicotinamide adenine dinucleotide), and 1 μM FMN (flavin mononucleotide) was introduced into the sample holder container from a syringe. The reaction substrate solution introduced into the sample holder container passed through the disk cartridge and was then collected in a collection container.

[0040] The relationship between the amount of reaction substrate solution sent from the syringe to the disk cartridge and the decolorization rate of the reaction substrate solution collected in the collection container is shown in Figure 7. In Figure 7, the evaluation results of a disk cartridge containing 36 mg of an enzyme complex with AzoR supported on mesoporous silica with a pore size of 4 nm are shown as "AzoR-CAPC filter (SBA4, 36 mg)," and the evaluation results of a disk cartridge containing 29 mg of an enzyme complex with AzoR supported on mesoporous silica with a pore size of 8 nm are shown as "AzoR-CAPC filter (SBA8, 29 mg)."

[0041] When the amount of the reaction substrate solution sent was 1 to 6 mL, the sending rate of the reaction substrate solution was 0.1 mL / min (1 mL was collected in 10 minutes), and when the amount of the reaction substrate solution sent was 7 to 15 mL, the sending rate of the reaction substrate solution was 0.0333 mL / min (1 mL was collected in 30 minutes). The decolorization rate was calculated by observing the change in absorbance at a wavelength of 430 nm. As shown in FIG. 7, it was found that methyl red was reductively decomposed (decolorized) using the disk cartridge of Example 1. In addition, the decolorization rate changed depending on the sending rate of the reaction substrate solution, that is, the time that the reaction substrate solution remained in the disk cartridge. Furthermore, the decolorization rate changed depending on the difference in the pore size of the mesoporous silica. These were similar to the reductive decomposition (decolorization) reaction of methyl red in a batch system using only mesoporous silica powder.

[0042] Comparative Example Except for the use of mesoporous silica not carrying AzoR, the relationship between the amount of reaction substrate solution sent from the syringe to the disk cartridge and the decolorization rate of the reaction substrate solution collected in the collection container was examined in the same manner as in Example 1. The results are shown in FIG. 8. In FIG. 8, the evaluation results of the disk cartridge containing 23 mg of SBA-15 type mesoporous silica with a pore diameter of 4 nm are shown as "CAPC filter (SBA4, 23 mg)", and the evaluation results of the disk cartridge containing 21 mg of SBA-15 type mesoporous silica with a pore diameter of 8 nm are shown as "AzoR-CAPC filter (SBA8, 21 mg)". As shown in FIG. 8, methyl red was hardly decolorized in the disk cartridge of the comparative example.

[0043] Example 2 (Disc cartridge manufacturing) A disk cartridge was produced by the method for producing an enzyme-encapsulated complex according to the second embodiment in the following procedure. A disk cartridge material encapsulating 25 mg of SBA-15 mesoporous silica with a pore size of 4 nm not carrying AzoR was produced in the same manner as in the comparative example. The disk cartridge material was set in the sample holder container of the evaluation device used in Example 1 in an incubator kept at a temperature of 30°C. 5 mL of 25 mM Tris-HCl buffer (pH 7.5) was fed to the disk cartridge material at a feeding rate of 0.1 mL / min.

[0044] In the same manner as in Example 1, a 0.25 mg / mL AzoR solution was prepared using 25 mM Tris-HCl buffer (pH 7.5). 4 mL of this AzoR solution was further fed to the disk cartridge material at a feed rate of 0.1 mL / min to obtain a disk cartridge precursor. 5 mL of 25 mM Tris-HCl buffer (pH 7.5) was fed to the disk cartridge precursor at a feed rate of 0.1 mL / min and washed to obtain a disk cartridge containing mesoporous silica carrying AzoR. In the same manner, a disk cartridge containing 23 mg of SBA-15 type mesoporous silica with a pore size of 8 nm carrying AzoR was obtained.

[0045] (evaluation) Except for the fact that the liquid delivery speed was fixed at 0.1 mL / min (1 mL was collected in 10 minutes), the same procedure as in Example 1 was used to evaluate whether a disk cartridge in which the enzyme functions could be prepared by decolorizing the azo dye. The relationship between the amount of reaction substrate solution delivered from the syringe to the disk cartridge and the decolorization rate of the reaction substrate solution collected in the collection container is shown in FIG. 9. In FIG. 9, the evaluation results of the disk cartridge containing an enzyme complex in which AzoR is supported on 25 mg of mesoporous silica with a pore size of 4 nm is shown as "AzoR-CAPC filter (SBA4, 25 mg)", and the evaluation results of the disk cartridge containing an enzyme complex in which AzoR is supported on 23 mg of mesoporous silica with a pore size of 8 nm is shown as "AzoR-CAPC filter (SBA8, 23 mg)".

[0046] As shown in Fig. 9, the disk cartridge of Example 2 showed a highly efficient reduction decomposition of methyl red with a decolorization rate of about 90%. This suggests that a disk cartridge in which the enzyme functions can be obtained by later loading an enzyme into the porous body of the disk cartridge material containing a porous body that does not support an enzyme. Also, as shown in Fig. 9, a tendency for the decolorization rate to change depending on the pore size of the mesoporous silica was observed, as in Example 1. [Explanation of symbols]

[0047] 10 Fibrous resin sheet 10a hole 10b Fibrous resin sheet having a hole formed in the center 10c Fibrous resin sheet with fibrous resin also present in the center 12 Lower mold 14 Upper mold 16 Gas flow path 18 First resin member 18a Recess 20 Fibrous resin sheet 22 Lower mold 24 Upper mold 24a Recess 26 Gas flow path 28 Second resin member 28a Convex 30 Enzyme Complexes 32 Lower mold 34 Upper mold 36 Gas flow path 40 Enzyme Inclusion Complex

Claims

1. A laminate of a plurality of fibrous resin sheets each having a hole formed in the center, and a first resin member in which a laminate of a plurality of fibrous resin sheets each having fibrous resin also in the center is overlapped and a recess due to the hole is formed in the center; An enzyme complex comprising a porous body and an enzyme supported on the porous body, the enzyme complex being accommodated in the recess; A second resin member including a plurality of laminated fibrous resin sheets and covering the enzyme complex; having The first resin member and the second resin member are joined at their peripheral portions, The mesh of the first resin member and the second resin member is larger than the enzyme and smaller than the enzyme complex.

2. In claim 1, The enzyme-encapsulating complex has a convex portion formed in the center of the second resin member, the convex portion corresponding to the concave portion.

3. In claim 1 or 2, The enzyme-encapsulating complex, wherein the porous body is mesoporous silica.

4. In any one of claims 1 to 3, The pore diameter of the porous body is 1 nm to 50 nm, The porous material has a particle size of 0.1 μm to 200 μm.

5. A method for producing an enzyme-encapsulated complex, in which an enzyme complex comprising a porous body and an enzyme supported on the porous body is encapsulated between a first resin member and a second resin member, comprising: the first resin member includes a plurality of laminated fibrous resin sheets and a mesh larger than the enzyme and smaller than the enzyme complex, and a recess is formed in the center; the second resin member includes a plurality of laminated fibrous resin sheets and a mesh larger than the enzyme and smaller than the enzyme complex; an enzyme complex disposing step of disposing the enzyme complex in the recess; an enzyme complex encapsulation step of covering the enzyme complex with the second resin member and bonding the peripheries of the first resin member and the second resin member to obtain the enzyme encapsulation complex; A method for producing an enzyme-encapsulating complex comprising the compound comprising:

6. A method for producing an enzyme-encapsulated complex, in which an enzyme complex comprising a porous body and an enzyme supported on the porous body is encapsulated between a first resin member and a second resin member, the first resin member includes a plurality of laminated fibrous resin sheets and a mesh larger than the enzyme and smaller than the porous body, and a recess is formed in the center; the second resin member includes a plurality of laminated fibrous resin sheets and a mesh larger than the enzyme and smaller than the porous body; a porous body disposing step of disposing the porous body in the recess; a porous body encapsulation step of covering the porous body with the second resin member, bonding peripheries of the first resin member and the second resin member to encapsulate the porous body between the first resin member and the second resin member; an enzyme carrying step of contacting a liquid containing the enzyme with the porous body after the porous body encapsulation step, thereby carrying the enzyme in the porous body to obtain the enzyme-encapsulated complex; A method for producing an enzyme-encapsulating complex comprising the compound comprising:

7. In claim 5 or 6, The method for producing an enzyme-encapsulated complex further includes a first resin member preparation step, which includes, before the enzyme complex arrangement step or the porous body arrangement step, a first plasticization step in which a plurality of fibrous resin sheets are stacked, and carbon dioxide in a liquid state or a high-density gas state is brought into contact with the plurality of fibrous resins to plasticize the surfaces of the plurality of fibrous resins, a first deformation step in which the plurality of fibrous resins with their plasticized surfaces are deformed by pressing, and a first fixation step in which the carbon dioxide is removed in a state in which the plurality of fibrous resins are deformed, and the plurality of fibrous resins are fixed to obtain the first resin member.

8. In claim 7, A method for producing an enzyme-encapsulating complex, in which the first plasticization process involves stacking a laminate of multiple fibrous resin sheets each having a hole formed in the center and a laminate of multiple fibrous resin sheets each having fibrous resin also present in the center, and contacting them with carbon dioxide in a liquid state or a high-density gaseous state.

9. In any one of claims 5 to 8, The method for producing an enzyme-encapsulated complex further includes a second resin member preparation process, which includes, prior to the enzyme complex encapsulation process or the porous body encapsulation process, stacking a plurality of fibrous resin sheets, bringing carbon dioxide in a liquid state or a high-density gaseous state into contact with the plurality of fibrous resins, and plasticizing the surfaces of the plurality of fibrous resins, a second deformation process deforming the plurality of fibrous resins whose surfaces have been plasticized by pressing, and a second fixation process removing the carbon dioxide in a state in which the plurality of fibrous resins are deformed, and fixing the plurality of fibrous resins to obtain the second resin member.

10. In claim 9, In the second deformation step, the plurality of fibrous resins are deformed by pressure so that a convex portion is formed in the center.

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