Fabrication method of enzyme immobilized zirconia

Abstract

The method for manufacturing lipase-immobilized zirconia according to the present invention comprises: a first step of mixing a lipase solution and a zirconia dispersion; a second step of separating the solid after the first step, adding a glutaraldehyde solution and mixing, and then separating the solid; a third step of separating the solid after the second step by adding a phosphate buffer solution and washing; and a fourth step of adding a tris(hydroxymethyl)aminomethane-hydrochloride buffer solution after the third step.

Description

Technology Field

[0001] The present invention relates to a method for manufacturing lipase-immobilized zirconia. Background Technology

[0002] Enzymes can be utilized in the fields of pharmacy and chemistry to synthesize or decompose substances. Enzymatic reactions are generally environmentally friendly methods characterized by high efficiency under mild reaction conditions. Furthermore, enzymes often provide stereoselectivity and regioselectivity, offering the advantage of distinguishing and synthesizing stereoisomers that are difficult to differentiate in traditional organic synthesis.

[0003] However, enzymes have limitations due to a narrow applicable temperature and pH range, and there is a problem in that they are easily inactivated due to denaturation if the active temperature or pH is exceeded. Additionally, there is a technical limitation in separating and reusing enzymes after a reaction.

[0004] To solve these problems, a method of introducing enzymes into mesoporous silica has been developed, but the mounting efficiency is still not high, and there is a problem of decreased enzyme activity upon repeated use.

[0005] Therefore, there is a need to develop enzyme immobilization methods that can increase reuse efficiency. Prior art literature

[0006] Republic of Korea Published Patent Application No. 10-2007-0118805, Japanese Published Patent Application No. 2024-507907, Japanese Published Patent Application No. 2023-549607 The problem to be solved

[0007] The objective of the present invention is to provide a method for manufacturing easily recyclable lipase-immobilized zirconia.

[0008] Another objective of the present invention is to provide a method for manufacturing lipase-immobilized zirconia with excellent storage stability. means of solving the problem

[0009] A method for manufacturing lipase-immobilized zirconia according to the present invention comprises a first step of mixing a lipase solution and a zirconia dispersion;

[0010] A second step of adding and mixing a glutaraldehyde solution after the first step above, and then separating the solids;

[0011] A third step of separating solids after washing by adding a phosphate buffer solution following the second step above; and

[0012] It includes a fourth step of adding a tris(hydroxymethyl)aminomethane-hydrochloride buffer solution after the third step.

[0013] In a method for manufacturing lipase-immobilized zirconia according to one embodiment of the present invention, the lipase solution may be characterized by being prepared by adding lipase to a phosphate buffer.

[0014] In a method for manufacturing lipase-immobilized zirconia according to one embodiment of the present invention, the phosphate buffer used in the lipase solution in the first step may be characterized by having a concentration of 10 to 50 mM.

[0015] In a method for manufacturing lipase-immobilized zirconia according to one embodiment of the present invention, the zirconia dispersion in the first step may be characterized by containing 3 to 15 mg of zirconia dispersion per 1 ml of water.

[0016] In a method for manufacturing lipase-immobilized zirconia according to one embodiment of the present invention, the lipase solution may be characterized by having 2 to 25 mg of lipase added per 1 ml of solvent.

[0017] In a method for manufacturing lipase-immobilized zirconia according to one embodiment of the present invention, the third step may be characterized by being repeated 2 to 10 times.

[0018] In a method for manufacturing lipase-immobilized zirconia according to one embodiment of the present invention, the phosphate buffer solution used in the third step may be characterized by having a concentration of 70 to 150 mM.

[0019] In a method for manufacturing lipase-immobilized zirconia according to one embodiment of the present invention, the zirconia has an average size of primary particles of 50 to 300 nm, and

[0020] It can be characterized by the average size of the secondary particles being 1 to 10 μm.

[0021] In a method for manufacturing lipase-immobilized zirconia according to one embodiment of the present invention, the zirconia may be characterized by having a specific surface area of ​​140 to 180 m² / g.

[0022] In a method for manufacturing lipase-immobilized zirconia according to one embodiment of the present invention, the zirconia may be characterized by having an average pore diameter of 3 to 6 nm. Effects of the invention

[0023] The method for manufacturing lipase-immobilized zirconia according to the present invention comprises: a first step of mixing a lipase solution and a zirconia dispersion; a second step of separating the solid after the first step, adding a glutaraldehyde solution and mixing, and then separating the solid; a third step of adding a phosphate buffer solution and washing after the second step, and then separating the solid; and a fourth step of adding a tris(hydroxymethyl)aminomethane-hydrochloride buffer solution after the third step; the lipase-immobilized zirconia manufactured by this method has the advantage of exhibiting high storage stability and high efficiency when reused. Brief explanation of the drawing

[0024] Figure 1 shows the results of observing zirconia used in the lipase-immobilized zirconia manufacturing method according to one embodiment of the present invention using TEM (Transmission Electron Microscope) and SEM (Scanning Electron Microscope). Figure 2 illustrates the results of analyzing pore characteristics through the adsorption and desorption of synthesized zirconia used in the method for manufacturing lipase-immobilized zirconia according to one embodiment of the present invention. Figure 3 illustrates the FT-IR (Fourier Transform Infrared Spectroscopy) and XRD (X-ray Diffraction) analysis results of synthesized zirconia used in the method for manufacturing lipase-immobilized zirconia according to one embodiment of the present invention. Figure 4 shows the storage stability of lipase-immobilized zirconia according to one embodiment of the present invention and illustrates the results. Figure 5 shows the efficiency of repeated use of lipase-immobilized zirconia according to one embodiment of the present invention and illustrates the results. Specific details for implementing the invention

[0025] The advantages and features of the embodiments of the present invention, and the methods for achieving them, will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims. Throughout the specification, the same reference numerals refer to the same components.

[0026] In describing the embodiments of the present invention, specific descriptions of known functions or configurations will be omitted if it is determined that such detailed descriptions could unnecessarily obscure the essence of the invention. Furthermore, the terms described below are defined in consideration of their functions in the embodiments of the present invention, and these definitions may vary depending on the intentions or practices of the user or operator. Therefore, such definitions should be based on the content throughout this specification.

[0027] A method for manufacturing lipase-immobilized zirconia according to the present invention comprises a first step of mixing a lipase solution and a zirconia dispersion;

[0028] A second step of separating the solids after the first step above, adding and mixing a glutaraldehyde solution, and then separating the solids;

[0029] A third step of separating solids after washing by adding a phosphate buffer solution following the second step above; and

[0030] It includes a fourth step of adding a tris(hydroxymethyl)aminomethane-hydrochloride buffer solution after the third step.

[0031] The method for manufacturing lipase-immobilized zirconia according to the present invention includes the first to fourth steps, and has the advantage of exhibiting excellent binding strength in which lipase is immobilized inside the zirconia not only through physical adsorption but also through a reaction with the addition of glutaraldehyde.

[0032] In the first step, the lipase solution may be prepared by adding lipase to a phosphate buffer, wherein the phosphate buffer may satisfy a concentration of 10 to 50 mM, preferably 15 to 40 mM, and the lipase may be added at a concentration of 2 to 25 mg, preferably 5 to 20 mg, and more preferably 10 to 15 mg per 1 ml of solvent. By satisfying these ranges for the lipase addition amount, a fast reaction rate and excellent lipase activity can be secured through high lipase loading.

[0033] In the first step, 3 to 15 mg of zirconia dispersion, preferably 7 to 13 mg, can be added per 1 ml of water, and within this range, the production efficiency of lipase-immobilized zirconia can be secured. In addition, the lipase solution and zirconia dispersion mixed in the first step can be mixed in a volume ratio of 1:0.8 to 1.3, and the highest lipase loading can be exhibited at this mixing ratio.

[0034] The method for manufacturing lipase-immobilized zirconia according to the present invention includes a second step of adding and mixing a glutaraldehyde solution after the first step and separating the solid components. However, to ensure uniform mixing of the zirconia dispersion and the lipase solution after the first step and before performing the second step, the method may further include steps of ultrasound, stirring, and centrifugation. At this time, ultrasound may be applied for 3 to 20 minutes, and preferably, the method may further include a step of stirring the mixture of the first step at 200 to 350 rpm after applying ultrasound. Subsequently, after separating only the solid components through centrifugation, the process of the second step may be carried out.

[0035] The second step involves introducing a glutaraldehyde solution to more firmly bind lipase inside the pores of the zirconia. The glutaraldehyde solution introduced at this time may be an aqueous solution with a concentration of 0.3 to 0.8 weight%, preferably 0.4 to 0.7 weight%. Additionally, 1.5 to 3 ml of the glutaraldehyde solution may be introduced relative to 1 ml of the lipase solution. Within this range, sufficient and rapid binding of lipase and zirconia can be ensured. More specifically, the second step may include a stirring process to ensure uniform and rapid dispersion of zirconia and lipase, and stirring may be performed at 100 to 200 rpm for 60 to 500 minutes. After stirring, only the solid components may be separated through centrifugation before performing the third step.

[0036] The third step involves separating solids after washing by adding a phosphate buffer solution following the second step. The phosphate buffer solution added at this time may have a concentration of 70 to 150 mM, preferably 80 to 120 mM, and washing efficiency can be improved within this range. Additionally, the washing in the third step may be repeated 2 to 10 times, preferably 2 to 6 times, and within this range, glutaraldehyde remaining in the micropores of zirconia can be removed, and high lipase immobilization efficiency can be achieved. Furthermore, the phosphate buffer solution used for washing may be added in an amount of at least 1 ml, preferably 1.5 ml to 5 ml, relative to 1 ml of the lipase solution to perform the washing. Additionally, filtering or centrifugation may be used for the separation of solids.

[0037] The method includes a fourth step of adding a Tris(hydroxymethyl)aminomethane-hydrochloride (Tris-HCl) buffer solution after the third step. The Tris-HCl buffer solution used in the fourth step may have a concentration of 70 to 150 mM, preferably 80 to 120 mM, and a pH of about 7.5 to 8.5. Additionally, after the fourth step, the method may further include a fifth step of washing again with a phosphate buffer solution, wherein the phosphate buffer solution may satisfy a concentration of 10 to 50 mM, preferably 15 to 40 mM.

[0038] The zirconia used in the first step is described in detail below. Zirconia can be synthesized by a hydrothermal synthesis method after mixing an aqueous solution of a zirconia precursor and an aqueous solution of a base. Specifically, zirconium (IV) oxynitrate hydrate (ZrO(NO3)2·H2O) can be used as the zirconia precursor, and the concentrations of the aqueous solution of the zirconia precursor and the aqueous solution of the base can each be 0.005 to 0.02 M. After mixing the aqueous solution of the zirconia precursor and the aqueous solution of the base, the process may include a step of heating with stirring at 100 to 150 °C for 6 to 30 hours to perform hydrothermal synthesis, and finally, zirconia can be synthesized by removing moisture from the solid after heating. The moisture can be removed through calcination treatment at 600 to 800 °C for 1 to 10 hours. Zirconia used for lipase immobilization can be synthesized by grinding the particles through milling after calcination.

[0039] Zirconia produced through this process has an average primary particle size of 50 to 300 nm, preferably 80 to 200 nm, and primary particles of this average particle size can aggregate to form secondary particles. The secondary particles of zirconia can satisfy an average size of 1 to 10 μm, preferably 1.5 to 5 μm. In addition, this zirconia satisfies a specific surface area of ​​140 to 180 m² / g, preferably 145 to 170 m² / g, and an average pore diameter of 3 to 6 nm, preferably 3.3 to 5 nm. Since the zirconia satisfies these specifications, lipase can be immobilized within the pores, and there is an advantage in that the lipase immobilization effect and a large contact area with the reactant during the reaction after immobilization can be secured due to the large specific surface area.

[0041] The present invention will be explained in detail below through examples and comparative examples. The following examples are intended only to aid in understanding the present invention, and the scope of the present invention is not limited by the following examples.

[0042] [Preparation Example 1]

[0043] 1. Preparation of porous zirconia

[0044] A 0.01 M zirconia precursor solution was prepared by dissolving Zirconium(IV) oxynitrate hydrate (ZrO(NO3)2·xH2O) in distilled water. NaOH was added to adjust the pH of the zirconia precursor solution to 11, and the solution was stirred at 120 °C for 12 hours using a hydrothermal synthesis method. The synthesis solution was filtrationed using an aspirator, washed with distilled water, and dried at 80 °C for 3 hours to produce ZrO(OH)2. This was then calcined in an electric furnace at 700 °C for 3 hours to produce ZrO2. After calcination, the solution was milled using a milling machine for 20 minutes to finally produce porous zirconia.

[0046] 2. Immobilization of lipase on zirconia

[0047] A 10 mg / ml zirconia dispersion was prepared by adding 10 mg of the previously prepared porous zirconia per 1 ml of water. Separately, a lipase solution was prepared by adding 4 mg of lipase per 1 ml of pH 7 phosphate buffer solution (concentration 20 mM). 1 ml each of the lipase solution and the zirconia dispersion were added to a reactor, sonicated for 10 seconds, and stirred at 250 rpm for 180 minutes. Afterward, the solid was separated by centrifugation at 13,000 rpm for 2 minutes, 2 ml of a 0.5 wt% glutaraldehyde solution was added to the separated solid, and the mixture was stirred at 200 rpm for 150 minutes. Subsequently, the solid was separated by centrifugation at 13,000 rpm for 2 minutes, the buffer was decanted, and 2 ml of 100 mM phosphate buffer (pH 7) was added. The process of centrifugation followed by the addition of phosphate buffer was repeated 3 times, after which the solid was separated by centrifugation at 13,000 rpm for 2 minutes. 2 ml of 100 mM Tris-HCl (pH 8) was added to the separated solid, stirred at 200 rpm for 30 minutes, and centrifuged at 13,000 rpm for 2 minutes. The buffer solution was removed, 2 ml of 100 mM phosphate buffer (pH 7) was added, and the process of centrifugation to remove the buffer solution was repeated 3 times. Afterward, the mixture was centrifuged at 13,000 rpm for 2 minutes, and 2 ml of 20 mM phosphate buffer was added for use in the experiment.

[0049] [Preparation Examples 2 to 8]

[0050] Lipase-immobilized zirconia was prepared by the same method as in Preparation Example 1, but with different concentrations of the zirconia dispersion and lipase solution as shown in Table 1 below.

[0051] Sample name Zirconia dispersion concentration (mg / ml) Lipase solution concentration (mg / ml) Sample 1 10 4 Sample 2 10 8 Sample 3 10 12 Sample 4 10 16 Sample 5 5 4 Sample 6 5 8 Sample 7 5 12 Sample 8 5 16

[0052] Analysis of manufactured zirconia

[0053] The synthesized zirconia particles before lipase immobilization were analyzed by TEM (left) and SEM (right), and the results are shown in Fig. 1.

[0054] Referring to Figure 1, it can be seen that the synthesized zirconia particles consist of primary particles with an average particle size of 50 to 150 nm and secondary particles formed by aggregating these primary particles with an average particle size of about 2.36 μm.

[0055] Figure 2 and Table 2 illustrate the analysis of pore characteristics through the adsorption and desorption of the synthesized zirconia. Referring to Figure 2 and Table 2, it can be seen that the synthesized zirconia exhibits porous characteristics with a high specific surface area, and has a pore size of approximately 4 nm, which is sufficient for lipase to enter.

[0057] Sample name Specific surface area (㎡ / g) Pore ​​volume (cm³ / g) Pore ​​size (nm) Zirconia 159.2724 0.1610 4.0441

[0059] Figure 3 shows the results of analyzing synthesized zirconia particles by FT-IR (a) and XRD (b). Referring to Figure 3a, the zirconia peak is at 480 cm⁻¹. -1 , 1000 cm -1 It can be confirmed that the crystal structure of zirconia is monoclinic ZrO2 by referring to Fig. 3b.

[0061] Zirconia analysis after lipase fixation

[0062] Table 3 shows that the activity of lipase (LP) immobilized within the particle pores of lipase-immobilized zirconia prepared according to the present invention was measured using the concentration of p-nitrophenol generated from the hydrolysis of 9 mg of 4-nitrophenyl acetate dissolved in 1 mL of acetonitrile. Specifically, the method required 1.485 mL of 20 mM phosphate buffer (pH 8) and 15 µL of 50 mM 4-nitrophenyl acetate as substrates, and the reaction was performed for 5 minutes, with the absorbance in the A400 region measured at 1-minute intervals. The measured absorbance is proportional to the concentration of p-nitrophenol, and the lipase activity, efficiency, and lipase loading amount were derived from the average of the slope values ​​of the absorbance measured per reaction time. Additionally, in Table 3, the activity value per unit lipase refers to the lipase activity value divided by the loading amount.

[0064] Sample name Lipase activity (mM / min) efficiency(%) Loading (wt%) Unit lipase activity value Sample 1 0.063 21.6 7.9 0.007975 Sample 2 0.049 36.4 22.6 0.002168 Sample 3 0.045 2.6 3.1 0.014516 Sample 4 0.050 62.2 49.9 0.001002 Sample 5 0.034 34.4 21.6 0.001574 Sample 6 0.027 17.1 21.5 0.001256 Sample 7 0.020 4.2 9.1 0.002198 Sample 8 0.038 3.2 9.3 0.004086

[0066] Referring to Table 3, it can be seen that the activity value per unit lipase was highest in sample 3, and additional experiments were subsequently conducted based on sample 3.

[0068] Check storage stability

[0069] Storage stability was measured based on sample 3, which exhibited the highest activity and efficiency among the samples, and the results are shown in Fig. 4. Additionally, activity upon repeated use was measured and is shown in Fig. 5. Specifically, to verify the storage stability of lipase, lipase activity was measured, and the lipase loaded into the pores of zirconia particles was washed 4 to 5 times in a 20 mM phosphate buffer solution (pH 8.0) and stored at room temperature. After storage, activity was measured again, and the samples were stored using the same method. Referring to Fig. 4, it can be seen that the lipase maintains more than 75% of its initial activity even after 40 days. Fig. 5 illustrates the change in activity due to repeated use, showing that it maintains more than 55% of its activity even after reuse more than 7 times.

Claims

Claim 1 A method for manufacturing lipase-immobilized zirconia comprising: a first step of mixing a lipase solution and a zirconia dispersion; a second step of adding and mixing a glutaraldehyde solution after the first step and separating the solids; a third step of adding a phosphate buffer solution after the second step, washing and separating the solids; and a fourth step of adding a tris(hydroxymethyl)aminomethane-hydrochloride buffer solution after the third step; wherein the zirconia satisfies a specific surface area of ​​145 to 170 m² / g and an average pore diameter of 3 to 6 nm, the lipase solution contains 10 to 15 mg of lipase per 1 ml of solvent, the zirconia dispersion contains 7 to 13 mg of zirconia per 1 ml of water, and the phosphate buffer solution has a concentration of 80 to 120 mM. Claim 2 A method for manufacturing lipase-immobilized zirconia according to claim 1, characterized in that the lipase solution is prepared by adding lipase to a phosphate buffer. Claim 3 A method for manufacturing lipase-immobilized zirconia according to claim 2, characterized in that the phosphate buffer used in the lipase solution in the first step has a concentration of 10 to 50 mM. Claim 4 delete Claim 5 delete Claim 6 A method for manufacturing lipase-immobilized zirconia according to claim 1, characterized in that the third step is repeated 2 to 10 times. Claim 7 delete Claim 8 A method for manufacturing lipase-immobilized zirconia according to claim 1, characterized in that the zirconia has an average size of primary particles of 50 to 300 nm and an average size of secondary particles of 1 to 10 μm. Claim 9 delete Claim 10 delete

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