Method for producing the composite and the composite

Abstract

To provide a method for effectively utilizing shell powder, and especially to provide a method for producing a composite containing calcium carbonate crystals and a composite containing calcium carbonate crystals.SOLUTION: A method for producing a composite containing biomineral powder and calcium carbonate crystals comprises a step of adding biomineral powder to an aqueous solution containing calcium carbonate and adjusted to a pH of 8.5 or more and 11 or less to grow calcium carbonate crystals on surfaces of the biomineral powder. The biomineral powder contains a carbonic anhydrase and an acidic protein that binds with calcium ions or calcium carbonate.SELECTED DRAWING: Figure 5

Description

Technical Field

[0001] The present invention relates to a method for producing a composite and a composite. More specifically, the present invention relates to a method for producing a composite containing biomineral powder and calcium carbonate crystals, and a composite containing biomineral powder and calcium carbonate crystals.

Background Art

[0002] Since shells such as pearl oysters, scallops, and oysters are industrial wastes that are discarded after use, efforts have already been made to effectively utilize shells in various industries. Shell powder is blended into pharmaceuticals and cosmetics, and calcium carbonate in shells is heated to calcium oxide and used in disinfectants and the like. However, since these are costly and have low effectiveness, the examples applied to actual shells are limited.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

[0005] Developing a method for effectively utilizing seashell powder, which is currently treated as industrial waste, is also beneficial from the perspective of Sustainable Development Goals (SDGs). Therefore, the inventors of this invention have been studying methods for effectively utilizing seashell powder, and have focused on its use in the production of calcium carbonate crystals.

[0006] In other words, the present invention aims to provide a method for effectively utilizing seashell powder, and more particularly, to provide a method for producing a composite containing calcium carbonate crystals and a composite containing calcium carbonate crystals. [Means for solving the problem]

[0007] The method for producing the composite according to the embodiment of the present invention is: A method for producing a composite containing biomineral powder and calcium carbonate crystals, The process involves adding biomineral powder to an aqueous solution containing calcium carbonate and adjusted to a pH of 8.5 or higher and 11 or lower, and growing calcium carbonate crystals on the surface of the biomineral powder. The biomineral powder contains carbonic anhydrase and an acidic protein that binds to calcium ions or calcium carbonate. This is a method for manufacturing the composite.

[0008] Furthermore, the composite according to the embodiment of the present invention is A composite comprising biomineral powder and calcium carbonate crystals, The biomineral powder contains carbonic anhydrase and an acidic protein that binds to calcium ions or calcium carbonate. The calcium carbonate crystals are bonded to the surface of the biomineral powder. It is a complex. [Effects of the Invention]

[0009] According to the composite manufacturing method and composite according to the embodiment of the present invention, a composite manufacturing method and a composite containing calcium carbonate crystals can be provided as a method for effectively utilizing seashell powder. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 is a graph showing the results of measuring the concentration of calcium carbonate in the seawater prepared in Examples 1-6 and Comparative Examples 1-6. [Figure 2] Figure 2 is a graph (calibration curve) showing the relationship between the turbidity of seawater and the concentration of calcium carbonate. [Figure 3] Figure 3 is a photograph of the calcium carbonate crystals prepared in Example 7 observed by a scanning electron microscope (SEM). [Figure 4] Figure 4 is a photograph of the portion surrounded by the white frame line in the photograph shown in Figure 3 magnified 5 times. [Figure 5] Figure 5 is a photograph of the calcium carbonate crystals prepared in Example 8 observed by a scanning electron microscope (SEM). [Figure 6] Figure 6 is a photograph of the portion surrounded by the white frame line in the photograph shown in Figure 5 magnified 3.3 times. [Figure 7] Figure 7 is a graph showing the change over time of the pH and CO2 concentration of the seawater prepared in Example 9. [Figure 8] Figure 8 is a graph showing the change over time of the pH and CO2 concentration of the seawater prepared in Comparative Example 7. [Figure 9] Figure 9 is a graph showing the results of measuring the carbonic anhydrase activity of the biomineral powder used in Example 2. [Figure 10] Figure 10 is a graph showing the results of repeatedly measuring the carbonic anhydrase activity of the biomineral powder used in Example 2. [Figure 11] Figure 11 is a graph showing the relative carbonic anhydrase activity when the biomineral powder prepared in Example 10 is treated at various temperatures.

Embodiments for Carrying Out the Invention

[0011] As described above, the inventors have repeatedly studied methods for effectively using shell powder that has conventionally been treated as industrial waste. Shells are a typical example of products by biomineralization in which organisms produce minerals. Therefore, the inventors focused on the function of the calcification-related proteins contained in the shells and considered using shell powder in the process of manufacturing calcium carbonate crystals.

[0012] Calcium carbonate is abundant in seawater, and methods for obtaining calcium carbonate crystals from seawater have been investigated. However, conventional methods for synthesizing calcium carbonate crystals from seawater have resulted in slow crystal growth rates due to various inhibiting factors. Consequently, individual crystal particles become small, making it difficult to recover the synthesized calcium carbonate crystals.

[0013] On the other hand, in biomineralization, in which living organisms produce minerals, it is known that organisms can efficiently synthesize very large calcium carbonate crystals (see Non-Patent Document 1). The present inventors applied this mechanism and conducted extensive research to develop a method for rapidly synthesizing large-sized calcium carbonate crystals on the order of micrometers or millimeters that are easily recoverable, and have completed the present invention.

[0014] (Method of manufacturing the composite) A method for producing a composite according to an embodiment of the present invention is a method for producing a composite comprising biomineral powder and calcium carbonate crystals, comprising the step of adding biomineral powder to an aqueous solution (hereinafter simply referred to as "aqueous solution") containing calcium carbonate and adjusted to a pH of 8.5 or higher and 11 or lower, and growing calcium carbonate crystals on the surface of the biomineral powder. Each component will be described in detail below.

[0015] The aforementioned aqueous solution is not particularly limited as long as it contains calcium carbonate and has a pH adjusted to 8.5 or higher and 11 or lower. Examples of aqueous solutions containing calcium carbonate include seawater, brine obtained by reducing the water content of seawater, and demagnesium seawater from which magnesium ions have been removed. These aqueous solutions containing calcium carbonate can be used after adjusting their pH to 8.5 or higher and 11 or lower. The aforementioned aqueous solution derived from seawater contains a large amount of calcium carbonate as a natural resource and is therefore preferable to use. Of course, any aqueous solution containing calcium carbonate other than those derived from seawater can be used. For example, an aqueous solution produced by blowing carbon dioxide into lime milk (a suspension of slaked lime (calcium hydroxide)) to generate light calcium carbonate can be used.

[0016] The pH of the aqueous solution should be adjusted to be between 8.5 and 11. If the pH of the aqueous solution is less than 8.5, the precipitation of calcium carbonate crystals takes a very long time and is inefficient. Furthermore, considering the case where seawater or a substance derived from seawater is used as the aqueous solution, it is difficult to adjust the pH of seawater to above 11, so the upper limit of the pH of the aqueous solution should be around 11. Furthermore, for example, when using demagnesium-free seawater as an aqueous solution, a higher pH is preferable, so a pH of 8.5 or higher is preferable.

[0017] The aqueous solution is preferably pH-adjusted with a polyamine or sodium hydroxide. For example, putrescine can be preferably used as the polyamine. By adjusting the pH with a polyamine, the pH of the aqueous solution can be set to about 11.

[0018] As mentioned above, conventional methods of synthesizing calcium carbonate crystals from seawater have resulted in heterogeneous nucleation occurring throughout the solution, yielding only very small particle-sized calcium carbonate crystals that are difficult to recover. In contrast, the composite manufacturing method according to the embodiment of the present invention uses biomineral powder, allowing for the rapid and large-scale synthesis of composites containing large-sized calcium carbonate crystals on the order of micrometers or millimeters, which can then be easily recovered.

[0019] According to the method for producing the composite according to an embodiment of the present invention, calcium carbonate crystals can be grown on the surface of the biomineral powder by adding biomineral powder to an aqueous solution containing calcium carbonate. The biomineral powder can contain carbonic anhydrase and an acidic protein that binds to calcium ions or calcium carbonate. Examples of the biomineral powder include the shells of mollusks, the skeletons of cnidarians (corals), the exoskeletons of crustaceans, the skeletons of echinoderms, coccoliths of algae, shells of foraminifera, shells of brachiopods, otoliths of vertebrates, and cystris of higher plants. Among these, a powder containing the nacreous layer, prismatic layer, or both of the nacreous and prismatic layers of the pearl oyster (Pinctada fucata) is preferred. The nacreous and prismatic layers of the pearl oyster contain nacrein as carbonic anhydrase and Pif as an acidic protein that binds to calcium ions or calcium carbonate.

[0020] As a biomineral powder, for example, it is preferable to use a powder made from the nacreous layer obtained by peeling off the outer layer (prismatic layer) of the Akoya oyster shell and collecting the nacreous layer. As mentioned above, the prismatic layer also contains carbonic anhydrase and acidic proteins that bind to calcium ions or calcium carbonate. For this reason, the prismatic layer itself or a mixture of the prismatic layer and the nacreous layer can also be preferably used. However, since the prismatic layer is brown and interferes with the measurement of carbonic anhydrase activity, in the examples described later, only the nacreous layer was powdered and used. The method for powdering the nacreous layer is not particularly limited, and for example, a pulverizer using a cutter or a ball mill can be used. There are no particular restrictions on the method for removing the prismatic layer; for example, methods such as scraping it off with a grinder can be employed. Alternatively, the outer layer (prismatic layer) and nacreous layer can be separated by treating the pearl oyster shell with sodium hypochlorite.

[0021] The amount of biomineral powder added to the aqueous solution is not particularly limited and can be adjusted as appropriate depending on the concentration of calcium carbonate contained in the aqueous solution. Increasing the amount of biomineral powder added can increase the amount of calcium carbonate crystals precipitated per unit time. For example, if the aqueous solution is made from seawater, it is preferable to add 0.1 mg or more per 25 mL of the aqueous solution.

[0022] The particle size of the biomineral powder added to the aqueous solution is not particularly limited. For example, particles of about 1 μm or larger are preferably used, and particles of about 1 μm or larger and 1 mm or smaller are more preferably used. When the particle size of the biomineral powder is 1 μm or larger, the cost incurred in reducing the particle size can be reduced, and problems when recovering the resulting composite (for example, clogging in the case of filters) can be less likely to occur, making handling easier. On the other hand, when the particle size of the biomineral powder is 1 mm or smaller, it is possible to suppress increased raw material costs and a decrease in the recovery efficiency of the composite (especially calcium carbonate crystals) due to a smaller specific surface area. From this viewpoint, the particle size of the biomineral powder should be appropriately adjusted depending on the system.

[0023] The carbonic anhydrase contained in the biomineral powder exhibits excellent thermal stability. If the carbonic anhydrase activity of the biomineral powder after heat treatment at 60°C for 2 hours is taken as 100%, it maintains approximately 90% or more activity at temperatures between 4°C and 70°C, approximately 70% or more activity at temperatures between 80°C and 100°C, and approximately 20% or more activity at temperatures between 110°C and 160°C. Therefore, the temperature of the aqueous solution that is maintained while adding biomineral powder to the aqueous solution and growing calcium carbonate crystals on the surface of the biomineral powder is not particularly limited, and can be, for example, between 4°C and 100°C. Considering the temperature range in which enzyme activity is maintained at a high level as described above, it is preferable to maintain the aqueous solution in a range of 4°C to 70°C. Furthermore, as mentioned above, the carbonic anhydrase contained in the biomineral powder has high heat resistance, so even when the pearl layer is processed at high speed during pulverization and exposed to high temperatures due to friction, the carbonic anhydrase activity can be maintained. Furthermore, the biomineral powder described above can also be applied to Carbon dioxide Capture and Storage (CCS), where carbon dioxide is adsorbed onto amines. In CCS, adding the biomineral powder to an aqueous amine solution shortens the reaction time due to the action of carbonic anhydrase. Moreover, even if heat treatment is performed to regenerate the amine, the activity of carbonic anhydrase contained in the biomineral powder is maintained, making it useful as a catalyst.

[0024] As described above, in the method for producing the composite according to the embodiment of the present invention, calcium carbonate crystals can be epitaxially grown on the surface of the biomineral powder by adding the biomineral powder to the aqueous solution. In particular, by using biomineral powder, we succeeded in obtaining large-sized calcium carbonate crystals. The size of the calcium carbonate crystals can be freely increased or decreased by adjusting the size of the biomineral powder.

[0025] Furthermore, as shown in the examples described later, the inventors have scientifically demonstrated that biomineral powder (seashell powder) possesses carbonic anhydrase activity and activity that induces the epitaxial growth of calcium carbonate crystals. They have also discovered for the first time that these properties of biomineral powder can be used in a business to fix and recover gaseous carbon dioxide in calcium carbonate crystals in an aqueous solution.

[0026] (complex) The composite according to the embodiment of the present invention is a composite comprising biomineral powder and calcium carbonate crystals. The biomineral powder is the same as that described in the method for producing the composite according to the embodiment of the present invention described above, and contains carbonic anhydrase and an acidic protein that binds to calcium ions or calcium carbonate. The calcium carbonate crystals are bound to the surface of the biomineral powder. Such composites containing calcium carbonate crystals can be used in a variety of fields, including cement materials, plastic reinforcing agents, food additives, and calcium fertilizers.

[0027] The following describes an embodiment of the method for producing the composite of the present invention. (1) A method for producing a composite containing biomineral powder and calcium carbonate crystals, The process involves adding biomineral powder to an aqueous solution containing calcium carbonate and adjusted to a pH of 8.5 or higher and 11 or lower, and growing calcium carbonate crystals on the surface of the biomineral powder. The biomineral powder contains carbonic anhydrase and an acidic protein that binds to calcium ions or calcium carbonate. A method for producing the composite. (2) The method for producing the composite according to (1) above, wherein the aqueous solution is seawater. (3) The method for producing the composite according to (1) or (2) above, wherein the aqueous solution has been pH-adjusted with a polyamine or sodium hydroxide. (4) The method for producing the complex described in (3) above, wherein the polyamine is putrescine. (5) The method for producing the composite according to any one of the above items (1) to (4), wherein the amount of biomineral powder added is 0.1 mg or more per 25 mL of the aqueous solution. (6) The method for producing the composite according to any one of the above items (1) to (5), wherein the biomineral powder has a particle size of 1 μm or more. (7) A method for producing the composite according to any one of the above items (1) to (6), wherein the biomineral powder comprises the nacreous layer, prismatic layer, or nacreous layer and prismatic layer of the Akoya oyster. (8) The method for producing the composite according to any one of the above items (1) to (7), wherein the biomineral powder is obtained by peeling the outer layer of the shell of an Akoya oyster to recover the nacreous layer and then pulverizing the nacreous layer. (9) The method for producing the composite according to any one of the above items (1) to (7), wherein the biomineral powder is obtained by removing the outer layer from the shell of an Akoya oyster by reacting it with sodium hypochlorite, recovering the nacreous layer, and then pulverizing the nacreous layer. (10) A method for producing a composite according to any one of the above items (1) to (9), wherein the step of growing the calcium carbonate crystals is carried out at a temperature of 4°C or higher and 70°C or lower.

[0028] The embodiments of the composite of the present invention are as follows. (11) A composite comprising biomineral powder and calcium carbonate crystals, The biomineral powder contains carbonic anhydrase and an acidic protein that binds to calcium ions or calcium carbonate. The calcium carbonate crystals are bonded to the surface of the biomineral powder. A complex. [Examples]

[0029] The present invention will be described in detail below with reference to examples, but the scope of the present invention is not limited to these examples.

[0030] (Example 1) -Preparation of biomineral powder- After washing the shells of Akoya oysters, the outer layer was removed using a grinder, and the nacreous layer, separated from the shell, was crushed in a ball mill to obtain a biomineral powder with a particle size (number average diameter) of approximately 9 μm. The particle size (number average diameter) of the biomineral powder was measured using a particle size distribution analyzer: HORIBA LA-300 manufactured by HORIBA, Ltd., under the conditions of relative refractive index: 1.18 and solvent: pure water (the same conditions apply to the following examples). -Manufacturing of the composite- Sodium hydroxide was added to filtered natural seawater to a concentration of 2 mmol / L. The pH of the seawater at this time was approximately 9.7. Natural seawater refers to seawater with a typical composition. 10 mg of the biomineral powder prepared above was added to 25 mL of seawater. The mixture was then stirred and left to stand at 25°C for at least 12 hours to allow calcium carbonate crystals to grow on the surface of the biomineral powder.

[0031] (Example 2) Biomineral powder was prepared in the same manner as in Example 1, except that the particle size (number average diameter) of the pearlescent powder was set to approximately 17 μm, and the composite was manufactured.

[0032] (Example 3) -Preparation of biomineral powder- Biomineral powder was prepared in the same manner as in Example 2. -Manufacturing of the composite- Seawater prepared in the same manner as in Example 1 was to which putrescine was added instead of sodium hydroxide to a concentration of 2 mmol / L. The pH of the seawater at this time was 9.7. 0.1 mg of the biomineral powder used in Example 2 was added to 25 mL of this seawater. The mixture was then stirred and left to stand at 25°C for more than 12 hours to allow calcium carbonate crystals to grow on the surface of the biomineral powder.

[0033] (Example 4) -Preparation of biomineral powder- Biomineral powder was prepared in the same manner as in Example 1. -Manufacturing of the composite- In Example 3, the composite was prepared in the same manner as in Example 3, except that 1 mg of the biomineral powder used in Example 1 was added to 25 mL of seawater.

[0034] (Example 5) -Preparation of biomineral powder- Biomineral powder was prepared in the same manner as in Example 1. -Manufacturing of the composite- In Example 3, the composite was prepared in the same manner as in Example 3, except that 10 mg of the biomineral powder used in Example 1 was added to 25 mL of seawater.

[0035] (Example 6) -Preparation of biomineral powder- Biomineral powder was prepared in the same manner as in Example 2. -Manufacturing of the composite- In Example 3, the composite was prepared in the same manner as in Example 3, except that 10 mg of the biomineral powder used in Example 2 was added to 25 mL of seawater.

[0036] (Example 7) -Preparation of biomineral powder- Biomineral powder was prepared in the same manner as in Example 2. -Manufacturing of the composite- 5.55 mg of CaCl2 (Wako, 95.0+%) was placed in a 5 mL tube, and then 1 mg of the biomineral powder used in Example 2 was added. The pH was adjusted to 9.0 with sodium hydroxide to obtain 5 mL of an 11.5 mM NaHCO3 solution. This NaHCO3 solution was added to the 5 mL tube containing the powder and suspended. The mixture was left to stand overnight at 25°C to produce the complex.

[0037] (Example 8) -Preparation of biomineral powder- Biomineral powder was prepared in the same manner as in Example 2. -Manufacturing of the composite- In Example 7, the composite was prepared in the same manner as in Example 7, except that sodium hydroxide was added to adjust the pH of the NaHCO3 solution to 10.0.

[0038] (Comparative Example 1) The composite was manufactured in the same manner as in Example 1, except that biomineral powder was not added.

[0039] (Comparative Example 2) The composite was prepared in the same manner as in Example 1, except that 10 mg of CaCO3 was added instead of biomineral powder.

[0040] (Comparative Example 3) The composite was manufactured in the same manner as in Example 3, except that biomineral powder was not added.

[0041] (Comparative Example 4) The composite was prepared in the same manner as in Example 3, except that 0.1 mg of CaCO3 was added instead of biomineral powder.

[0042] (Comparative Example 5) The composite was prepared in the same manner as in Example 3, except that 1 mg of CaCO3 was added instead of the biomineral powder.

[0043] (Comparative Example 6) The composite was prepared in the same manner as in Example 3, except that 10 mg of CaCO3 was added instead of biomineral powder.

[0044] [evaluation] -Measuring turbidity- Each seawater containing the composites prepared in Examples 1-6 and Comparative Examples 1-6 was stirred by pipetting, and the results were measured using a spectrophotometer (Thermo Scientific, NanoDrop One). CThe concentration of calcium carbonate crystals was calculated by measuring the turbidity (OD600) of seawater. The results are shown in Figure 1. The concentration of calcium carbonate crystals was calculated using a calibration curve (see Figure 2) created by measuring the turbidity (OD600) of seawater with a known concentration of calcium carbonate crystals. The results shown in Figure 1 were calculated by subtracting the turbidity of each seawater sample, which was left to stand overnight with biomineral powder or CaCO3 powder added and no base added, as background. Therefore, these results represent only the amount of calcium carbonate crystals precipitated during standing for 12 hours or more. Furthermore, when measuring turbidity, the pH of the seawater prepared in Examples 1-6 was approximately 8.3.

[0045] As shown in Figure 1, seawater to which biomineral powder was added (Examples 1-6) produced significantly more calcium carbonate than seawater without biomineral powder (Comparative Examples 1 and 3) or seawater with CaCO3 powder added (Comparative Examples 2, 4-6). The negative evaluation of calcium carbonate crystal production in Comparative Examples 5 and 6 is thought to be because the size of the produced calcium carbonate crystals was too small and they adhered to the walls of the sample bottles (glass bottles) (resulting in loss).

[0046] - Observation of the complex using SEM - In Examples 7 and 8, the precipitate of the complex containing calcium carbonate crystals formed at the bottom of the 5 mL tube was collected by centrifugation at 10,000 g for 5 minutes or more. The collected complex was washed three times with milli-Q water and stored in 100% ethanol. The precipitates in ethanol were then dried and observed using a scanning electron microscope (SEM, Hitachi, S-4800). Each dried composite was placed on an aluminum sample stage and coated with Pt / Pd using an ion coater (Hitachi, E-1030) for 60 seconds.

[0047] Figure 3 shows the results of observing the composite containing calcium carbonate crystals recovered in Example 7 using SEM (photograph). Figure 4 shows a 5x magnification of the area enclosed by the white frame in Figure 3. Similarly, Figure 5 shows the results of observing the composite containing calcium carbonate crystals recovered in Example 8 using SEM (photograph), and Figure 6 shows the area enclosed by the white frame in Figure 5 magnified 3.3 times. As shown in Figures 3 and 5, calcium carbonate crystals preferentially formed around the biomineral powder. Furthermore, when the initial pH was increased (Example 8), even more calcium carbonate crystals were generated (see Figures 5 and 6).

[0048] (Example 9) In a 1L beaker, 200mL of natural seawater and 500mg of biomineral powder were added, and then putrescine was added to a concentration of 10 mmol / L. The biomineral powder used was the one prepared in Example 2. The pH and CO2 concentration of this seawater were measured over a 72-hour period. A HORIBA F-72 benchtop pH meter was used to measure pH, and a K-Engineering OxyGuard CO2 dissolved carbon dioxide meter was used to measure CO2 concentration. The results are shown in Figure 7.

[0049] (Comparative Example 7) The pH and CO2 concentration of seawater prepared in the same manner as in Example 9, except that biomineral powder was not added, were measured over a 72-hour period. The results are shown in Figure 8.

[0050] As shown in Figures 7 and 8, the seawater in Example 9, to which biomineral powder was added, showed a decrease in pH at an earlier stage compared to the seawater in Comparative Example 7, to which biomineral powder was not added. Specifically, the time it took for the pH to drop by 0.5 from the initial pH (maximum pH) was 0.7 hours (pH 10.189 → pH 9.689) for the seawater in Example 9, compared to 2.5 hours (pH 10.101 → pH 9.601) for the seawater in Comparative Example 7. Since the decrease in pH is due to the formation of calcium carbonate crystals, it is thought that the biomineral powder promotes calcification (crystal growth) in the initial stages of the reaction.

[0051] -Measurement of carbonic anhydrase activity of biomineral powder- p-nitrophenyl acetate (p-NPA) was dissolved in acetonitrile solution to a concentration of 0.1 M. 0.72 ml of 50 mM Tris-HCl (pH 8.0) and 0.1 ml of 1 mM ZnSO4 solution were added to a 1.5 ml tube and mixed. 0.1 M p-NPA solution was then added to this mixture to a total volume of 1 ml, preparing solutions of each substrate concentration. 50 mg of the biomineral powder used in Example 2 was added to this substrate solution, and the activity of carbonic anhydrase was measured by measuring the absorbance at a wavelength of 400 nm. The results are shown in Figure 9.

[0052] Furthermore, the biomineral powder whose enzyme activity was measured as described above was collected and washed three times with a buffer solution of the following composition. The biomineral powder after washing was collected and its carbonic anhydrase activity was measured again using the method described above. buffer solution 50 mM Tris-HCl (pH 8.0) 100 μM ZnSO4 This procedure was repeated eight times. Using the carbonic anhydrase activity of the biomineral powder from the first trial as a baseline, the relative carbonic anhydrase activity of the biomineral powder from each trial is shown in Figure 10. As shown in Figure 10, the biomineral powder maintained its carbonic anhydrase activity even after repeated use, demonstrating its reusability as a functional bead.

[0053] (Example 10) Biomineral powders were prepared as follows, and their thermal stability was evaluated. -Preparation of biomineral powder- After washing the shells of the Akoya oysters, they were immersed in 30% sodium hypochlorite and stirred at 4°C for one week to remove the outer layer. The nacreous layer obtained after removing the outer layer was washed with distilled water to completely remove the sodium hypochlorite. Then, the nacreous layer was pulverized using a pulverizer (Wonder Crusher WC-3, manufactured by Osaka Chemical Co., Ltd.) to obtain biomineral powder. The particle size (average particle diameter) of the obtained biomineral powder was measured in the same manner as in Example 1 and was found to be 25.4 μm. - Evaluation of thermal stability - 50 mg of biomineral powder was left to stand at a constant temperature (4°C to 160°C) for 2 hours. Afterward, the samples were heated or cooled to 25°C, and the carbonic anhydrase activity of each biomineral powder was measured. The carbonic anhydrase activity was measured in the same manner as described above. The results are shown in Figure 11. Figure 11 shows the relative carbonic anhydrase activity, with the enzyme reaction rate at 60°C set to 100%. As shown in Figure 11, it was found that the biomineral powder maintained 100% carbonic anhydrase activity even when treated at 70°C. Furthermore, it maintained 70% carbonic anhydrase activity even when treated at 100°C, indicating that it maintained approximately 20% or more carbonic anhydrase activity even when treated at temperatures exceeding 100°C.

Claims

1. A method for producing a composite containing biomineral powder and calcium carbonate crystals, The process involves adding biomineral powder to an aqueous solution containing calcium carbonate and adjusted to a pH of 8.5 or higher and 11 or lower, and growing calcium carbonate crystals on the surface of the biomineral powder. The biomineral powder contains carbonic anhydrase and an acidic protein that binds to calcium ions or calcium carbonate. A method for producing the composite.

2. The method for producing the composite according to claim 1, wherein the aqueous solution is seawater.

3. The method for producing the composite according to claim 1 or 2, wherein the aqueous solution has been pH-adjusted with a polyamine or sodium hydroxide.

4. The method for producing the complex according to claim 3, wherein the polyamine is putrescine.

5. The method for producing the composite according to claim 1 or 2, wherein the amount of biomineral powder added is 0.1 mg or more per 25 mL of the aqueous solution.

6. The method for producing the composite according to claim 1 or 2, wherein the biomineral powder has a particle size of 1 μm or more.

7. The method for producing the composite according to claim 1 or 2, wherein the biomineral powder comprises the nacreous layer, prismatic layer, or both of the nacreous and prismatic layers of the Akoya oyster.

8. The method for producing the composite according to claim 1 or 2, wherein the biomineral powder is obtained by peeling the outer layer of the shell of an Akoya oyster, recovering the nacreous layer, and then pulverizing the nacreous layer.

9. The method for producing the composite according to claim 1 or 2, wherein the biomineral powder is obtained by removing the outer layer from the shell of an Akoya oyster by reacting it with sodium hypochlorite, recovering the nacreous layer, and then pulverizing the nacreous layer.

10. The method for producing the composite according to claim 1 or 2, wherein the step of growing the calcium carbonate crystals is carried out at a temperature of 4°C or higher and 70°C or lower.

11. A composite comprising biomineral powder and calcium carbonate crystals, The biomineral powder contains carbonic anhydrase and an acidic protein that binds to calcium ions or calcium carbonate. The calcium carbonate crystals are bonded to the surface of the biomineral powder. A complex.

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