KR102233145B1 - Composites formed by covalent bonding between hydroxyapatite and hydrophobic protein, and method for preparing the same - Google Patents

Abstract

The present invention provides a bioconjugation complex formed by covalently bonding a linker compound including a calcium phosphate-based bioceramic, an amino group and a carboxylate group, and a hydrophobic protein, and a method for preparing the same. That is, the present invention provides an effective manufacturing method for covalently conjugating a bioceramic hydroxyapatite and a protein by using a peptide-coupling agent.
A peptide-coupling agent can be used to directly conjugate a protein other than a hydrophobic protein to a bioceramic, and in the case of a hydrophobic protein, a bioceramic and a linker, or a bioceramic and linker conjugate and a biocatalyst can be combined by using a peptide-coupling agent. Bonding efficiency can be improved. In addition, by using a linker compound, a hydrophobic biocatalyst can be easily bonded to the polar bioceramic surface. The bioconjugation complex prepared by the above manufacturing method is covalently bonded to the bioceramic surface of functional proteins including hydrophobic proteins, so it can be applied to a support for tissue engineering suitable for a living body, a material for inducing and shielding tissue regeneration, and a biological cell proliferation material. have.

Description

Bioconjugation complex in which a protein is covalently bonded to hydroxyapatite, and a manufacturing method thereof {COMPOSITES FORMED BY COVALENT BONDING BETWEEN HYDROXYAPATITE AND HYDROPHOBIC PROTEIN, AND METHOD FOR PREPARING THE SAME}

The present invention relates to a bioconjugation complex in which a bioceramic (hydroxyapatite) and a hydrophobic protein are covalently bonded and a method for preparing the same, and more specifically, a linker compound and protein comprising a calcium phosphate bioceramic, an amino group and a carboxylate group It relates to a bioconjugation complex formed by covalent bonding and a method for preparing the same.

A representative ceramic used as a material for a support for regenerating hard tissues that connects tissues and tissues to regenerate tissues that have lost their self-healing function is calcium phosphate-based ceramics having chemical similarities to bone mineral phases. Hydroxyapatite (HAp, hydroxyapatite), one of the calcium phosphate-based ceramics, is a major component of the biological bone system and is considered to be biocompatible and environmentally benign. Hydroxyapatite is chemically composed of calcium ions, phosphates and hydroxides in the form of Ca ₅ (PO ₄ ) ₃ (OH) and can be easily synthesized in a test tube. Although the exact mechanism in the body is not clearly elucidated, osteoblasts play a role in producing hydroxyapatite in the growing bone. During bone growth, many proteins interact and bind with hydroxyapatite to assemble complex bone systems. In addition to the biocompatibility of hydroxyapatite, its native affinity for proteins has been used in many biological applications such as protein purification and nanomedicine. These properties of hydroxyapatite suggest that hydroxyapatite is suitable as a support material for immobilizing a biocatalyst. Although many proteins can be physically adsorbed on hydroxyapatite, proteins with hydrophobic surfaces cannot be adsorbed on the hydroxyapatite surface because the hydroxyapatite surface is charged and completely polarized. For conjugation of these hydrophobic proteins, covalent bonding would be an alternative approach.

Conventionally, as a method for conjugating a protein to hydroxyapatite, there is a covalent bonding method using a specific compound such as a bisphosphonate derived from hydrazine or amine as a linker. In the bisphosphonate linker compound derived from the hydrazine or amine, the phosphate group is coordinarily bonded to the calcium ion on the hydroxyapatite, and the other side of the linker compound (i.e., hydrazine or amine group) provides a site for protein binding, and thus hydroxyapatite and Conjugate proteins. However, the conjugation process using a bisphosphonate derived from hydrazine or amine as described above has a disadvantage in that a multi-step synthesis of a linker compound or additional modification of a protein is required.

There is also a method of using glycine as a linker compound, in which glycine is introduced into hydroxyapatite in the manufacturing process of hydroxyapatite, and then the amino group of glycine is used for covalent bonding to conjugate hydroxyapatite and protein. I wanted to. This method does not require multi-step synthesis of the linker compound, but has a disadvantage in that it is difficult to apply to previously prepared hydroxyapatite because glycine must be incorporated during the synthesis of hydroxyapatite.

Therefore, in order to broaden the application range of hydroxyapatite to proteins, particularly hydrophobic proteins, a simple protein conjugation process through covalent bonding is required.

Accordingly, the present inventors overcome the problems of the prior art, and as a result of intensive research efforts, a method for conjugating a bioceramic such as hydroxyapatite and a biocatalyst such as lipase, a hydrophobic protein, a series of peptide-coupling agents It was confirmed that apatite and protein can be directly covalently bonded. In particular, in the case of a hydrophobic protein, it was confirmed that a linker compound was required to cancel the repulsion between hydroxyapatite and the hydrophobic protein. Through this, in the case of a bioconjugation complex formed by covalently bonding a linker compound containing calcium phosphate-based bioceramics, an amino group and a carboxylate group, and a hydrophobic protein, a biocatalyst, a hydrophobic protein, is bound to the bioceramic by a linker compound. The possibility as an engineering scaffold or bone tissue scaffold was confirmed, and the present invention was completed.

KR 10-2015-0028976 A KR 10-2014-0028924 A

The main object of the present invention is to provide a bioconjugation complex in which a bioceramic and a hydrophobic protein are covalently bound and useful as a tissue engineering scaffold or bone tissue scaffold, and a method for preparing the same.

Another object of the present invention is to provide a tissue engineering scaffold using a bioconjugation complex to which the protein is covalently bonded, a medical material for bone filling or dental filling, and a membrane for inducing tissue regeneration or for shielding.

According to one aspect of the present invention, the present invention provides a bioconjugation complex to which the following three components are covalently bonded.

(i) calcium phosphate-based bioceramics;

(ii) a linker compound containing an amino group and a carboxylate group; And

(iii) hydrophobic protein.

Conventionally, since calcium phosphate-based bioceramics have a polar surface, it is difficult to bond a hydrophobic biocatalyst to the bioceramic surface, so there is a limit to use as a support for tissue engineering. Accordingly, the present inventors discovered that a hydrophobic biocatalyst can be easily bonded to the surface of a bioceramic, and the present invention was completed.

In the bioconjugation composite of the present invention, the calcium phosphate-based bioceramic may be any calcium phosphate-based bioceramic conventionally used to prepare a tissue engineering scaffold, but preferably, hydroxyapatite, At least one calcium phosphate selected from the group consisting of dicalcium phosphate dihydrate (DCPD), monocalcium phosphate monohydrate (MCPM), dicalcium phosphate anhydrous (DCPA), α-Tricalcium phosphate (α-TCP), and β-Tricalcium phosphate (β-TCP) It may be a biological ceramic, and more preferably, it is characterized in that it is hydroxyapatite (Hydroxyapatite).

In the bioconjugation complex of the present invention, the amino group of the linker compound is covalently bonded to the calcium phosphate-based bioceramic, and the carboxylate group of the linker compound is covalently bonded to the hydrophobic protein.

According to an embodiment of the present invention, the linker compound may be 6-aminohexanoic acid (6-AmH), and the 6-AmH has 5 carbons between an amino group and a carboxylate group. Because of this, it can help reduce the repulsive action between the surface of hydroxyapatite and the hydrophobic protein.

In the bioconjugation complex of the present invention, the hydrophobic protein may include any hydrophobic protein conventionally used as a biocatalyst, but is preferably a lipase.

In the bioconjugation complex of the present invention, the lipase is at least one selected from the group consisting of Candida lugosa lipase (CRL), Burkholderia sepacia lipase (BCL), and Candida antarctica lipase B (CAL-B). It is characterized by being a lipase.

According to an embodiment of the present invention, as a result of analyzing a scanning electron microscope (SEM) and powder X-ray diffraction (PXRD) to confirm the characteristics of the bioconjugation complex according to the present invention, the linker and the lipase bonded hydride It was confirmed that the morphology and crystallinity of the loxiapatite did not change (see Experimental Example 4, FIGS. 5 and 6). In addition, as a result of confirming the peak of hydroxyapatite to which the linker and lipase are bound through IR analysis, it was confirmed that all characteristic peaks for the linker, lipase, and hydroxyapatite were preserved (see Experimental Example 4 and Fig. 7). These results suggest that the bioconjugation composite according to the present invention is formed in a structure in which a linker and a biocatalyst are sequentially conjugated to a calcium phosphate-based bioceramic, so that it can be usefully used as a support for tissue engineering.

According to an embodiment of the present invention, as a result of confirming whether or not the lipase for the bioconjugation complex according to the present invention is recycled, it was confirmed that the bioconjugation complex to which the lipase is bound retains its activity even after 10 times of recycling. These results suggest that the bioconjugation complex according to the present invention can effectively maintain the stability of the binding protein, so that advantageous effects can be obtained when used as a tissue engineering scaffold (see Experimental Example 6 and Fig. 8).

According to another aspect of the present invention, the present invention provides a first step of activating by mixing a calcium phosphate-based ceramic with a peptide-coupling agent; A second step of suspending the calcium phosphate-based ceramic activated in the first step in the linker compound solution; A third step of centrifuging the suspension of the second step to obtain a precipitate, and then washing the precipitate; A fourth step of mixing and culturing the precipitate of the third step with a peptide-coupling agent; A fifth step of resuspending the precipitate of the fourth step in a solution containing a hydrophobic protein and then culturing it; And centrifuging the solution of the fifth step to obtain a complex to which a hydrophobic protein is bound.

Conventionally, since calcium phosphate-based bioceramics have a polar surface, it is difficult to bond a hydrophobic biocatalyst to the bioceramic surface, so there is a limit to use as a support for tissue engineering. Accordingly, the inventors of the present invention have found that when a peptide-coupling agent is used, the conjugation efficiency can be improved, and when a linker is used, a hydrophobic biocatalyst can be easily bonded to the bioceramic surface, and the present invention has been completed.

In the method of manufacturing the bioconjugation composite of the present invention, the calcium phosphate-based bioceramic in the first step may be any calcium phosphate-based bioceramic conventionally used to prepare a tissue engineering scaffold, but preferably Is composed of hydroxyapatite, DCPD (Dicalcium phosphate dihydrate), MCPM (Monocalcium phosphate monohydrate), DCPA (Dicalcium phosphate anhydrous), α-TCP (α-Tricalcium phosphate) and β-TCP (β-Tricalcium phosphate). It may be one or more calcium phosphate-based bioceramics selected from the group, and more preferably, it is characterized in that it is hydroxyapatite.

In the method for preparing the bioconjugation complex of the present invention, the peptide-coupling agent in the first step or the fourth step may be a carbodiimide-based coupling agent, for example, 1,1'- Carbonyldiimidazole (CDI), bis-Trichloromethylcarbonate (BTC), 2-(1H-Benzotriazol-1-yl)-N,N,N',N'-tetramethylaminium tetrafluoroborate (TBTU), bis-Trichloromethylcarbonate, 2-(1H-Benzotriazol -1-yl)-N,N,N',N'-tetramethylaminium hexafluorophosphate (HBTU), 2-(6-Chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylaminium hexafluorophosphate (HCTU), N-[(5-Chloro-1H-benzotriazol-1-yl)-dimethylamino-morpholino]-uronium hexafluorophosphate N-oxide (HDMC), preferably N,N'-dicyclohexyl carbodiimide (DCC ), N,N'-diisopropylcarbodiimide (DIC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

According to an embodiment of the present invention, in order to confirm the conjugation efficiency according to the peptide-coupling agent, when lipase is conjugated with a linker after activating a linker-bioceramic conjugate using three peptide-coupling agents, conjugation It was confirmed that the efficiency was increased, and in particular, when EDC was used as a peptide-coupling agent, it was confirmed that the conjugation efficiency was significantly higher than that when using other peptide-coupling agents (see Experimental Example 4 and FIG. 4). These results indicate that activation by the peptide-coupling agent is a factor affecting the conjugation efficiency of the linker and lipase, and is an essential component for preparing the bioconjugation complex according to the present invention, and EDC conjugates lipase to the linker. It suggests that it is most suitable as a peptide-coupling agent for

In the method for preparing the complex for conjugation of the present invention, the linker compound of the second step is characterized in that it is a compound containing an amino group and a carboxylate group.

According to an embodiment of the present invention, the linker compound may be any compound having an amino group and a carboxylic acid group, but preferably 3-aminopropanoic acid, 4-aminobutanoic acid ( 4-aminobutanoic acid), 5-Aminopentanoic acid, 6-aminohexanoic acid, 7-Aminoheptanoic acid, 8-aminooctanoic acid (8- It may be one or more compounds selected from the group consisting of aminooctanoic acid) and 9-aminononanoic acid, more preferably 6-aminohexanoic acid (6-AmH). It is done. Since the 6-AmH has 5 carbons between the amino group and the carboxylate group, it may help to reduce the repulsive action between the surface of hydroxyapatite and lipase. However, in the case of a non-hydrophobic protein, the protein and hydroxyapatite can be directly conjugated with a peptide-coupling agent without such a linker compound.

In the method for preparing the bioconjugation complex of the present invention, the hydrophobic protein in the fifth step is a lipase, preferably Candida lugosa lipase (CRL), Burgholderia sepacia lipase (BCL), and Candida. It is characterized in that it is at least one lipase selected from the group consisting of antarctica lipase B (CAL-B).

According to another aspect of the present invention, the present invention provides an industrial biocatalyst comprising a biocatalyst composite prepared by the above manufacturing method.

According to another aspect of the present invention, the present invention provides a tissue engineering scaffold comprising a bioconjugation complex prepared by the above manufacturing method.

According to another aspect of the present invention, the present invention provides a medical material for bone filling or dental cavity filling, including the bioconjugation composite prepared by the above manufacturing method.

According to another aspect of the present invention, the present invention provides a membrane for inducing tissue regeneration or for shielding comprising a bioconjugated composite prepared by the above manufacturing method.

The bioconjugation complex according to the present invention is for effectively regenerating tissue when organs or tissues in the human body lose or lose function, and a bioconjugation complex is prepared using a bioceramic, a linker, and a biocatalyst, The prepared bioconjugation complex can be applied to a tissue engineering scaffold, a material for inducing tissue regeneration and a material for tea, and a biological cell proliferation material.

According to another aspect of the present invention, the present invention comprises a calcium phosphate-based bioceramic by means of a peptide-coupling agent; And a complex for bioconjugation to which a functional protein is covalently bonded.

As described above, the method for preparing a bioconjugation complex according to the present invention improves the conjugation efficiency of a bioceramic and a linker or a bioceramic and a linker conjugate and a biocatalyst by using a peptide-coupling agent, and polarity by using a linker. The hydrophobic biocatalyst can be easily bonded to the phosphorus bioceramic surface. However, if it is not a hydrophobic protein, the protein and the bioceramic can be directly bonded without the help of a linker.

In addition, the bioconjugation complex prepared by the above manufacturing method is applied to a support for tissue engineering suitable for a living body, a material for inducing and shielding tissue regeneration, and a biological cell proliferation material, as a hydrophobic biocatalyst is bound to the surface of the bioceramic by a linker. I can.

1 is a diagram schematically illustrating a step of preparing a green fluorescent protein-conjugated-hydroxyapatite according to Example 1. FIG.
2 is a diagram showing the results according to Experimental Example 2 of the present invention (a: DCC, b: DIC, c: EDC were used as a peptide-coupling agent)
3 is a diagram showing the results according to Experimental Example 3 of the present invention.
4 is a diagram showing the results according to Experimental Example 4 of the present invention.
5 is a diagram showing SEM measurement results in Experimental Example 4 of the present invention.
6 is a diagram showing a PXRD measurement result in Experimental Example 4 of the present invention.
7 is a diagram showing the results of IR analysis in Experimental Example 4 of the present invention.
8 is a diagram showing the results according to Experimental Example 5 of the present invention.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are for illustrative purposes only, the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Preparation of green fluorescent protein-conjugated-hydroxyapatite

Three peptide couples such as N,N'-dicyclohexyl carbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to activate the pendant phosphate group Using a ring reagent, a contact process was performed through two steps, and a schematic diagram is shown in FIG. 1. The specific experimental method is as follows. First, hydroxyapatite (200mg) was immersed in a peptide coupling reagent (4 mL of 37.5 mM, DCC and DIC in methylene chloride or EDC in 100 mM MES buffer, pH 5.0) and shaken at 25° C. for 20 minutes. After sequentially washing with an appropriate solvent, the activated hydroxyapatite was suspended in an EGFP solution (1 mL, 1.55 mg/ml in pH 7.2 BES buffer) and incubated at 4° C. for 16 hours. Then, after washing three times with BES buffer (5 mM, pH 7.2), the prepared EGFP-conjugated-hydroxyapatite was dried in air.

Experimental Example 1: Measurement of the amount of EGFP conjugated to hydroxyapatite

The binding amount of EGFP bound to hydroxyapatite (6.9-8.6 mg/g of oxidized apatite) was calculated by subtracting the concentration of the conjugate and the conjugate before conjugation, and the results are shown in Table 1. The concentration of the EGFP solution was measured at 280 nm using ε = 22,015 M-1·cm ^-1 .

Coupling reagent EGFP binding amount (mg/g of hydroxyapatite) Not used 4.0 ± 0.05 DCC 6.9 ± 0.1 DIC 8.2 ± 0.03 EDC 8.6 ± 0.03

DIC> DCC)이 변하는 것을 확인하였다.As a result, as can be seen in Table 1 above, the peptide coupling reagent plays the same role, but the conjugation yield (EDC

DIC> DCC) was confirmed to change.

Experimental Example 2: Confirmation of conjugation properties of EGFP conjugated to hydroxyapatite

When considering EGFP conjugation, since it is known that proteins can be adsorbed to hydroxyapatite, EGFP may be physically adsorbed rather than covalently bonded, so after suspending hydroxyapatite in the EGFP solution without a binding reagent, the supernatant By measuring the concentration of EGFP, the amount of physically adsorbed or weakly coordinated was confirmed, and the EGFP-conjugated-hydroxyapatite phase was confirmed with a fluorescence microscope. The results are shown in Figs. 2(a to c).

As a result, it was confirmed that about 4 mg of EGFP was physically adsorbed to 1 g of hydroxyapatite, as can be seen in FIGS. 2 (a to c). This indicated that a certain amount of EGFP was physically adsorbed onto the hydroxyapatite.

Comparative Example 1: Preparation of hydroxyapatite-conjugated-lipase through direct conjugation

Candida rugosa lipase (CRL), Burkholderia cepacia lipase (BCL), and Candida antarctica lipase B (CAL-B) were used as lipases. First, in the same manner as in Example 1, Candida lugosa lipase (CRL) was directly conjugated to hydroxyapatite. CRLs were conjugated to hydroxyapatite after activation by DCC, DIC or EDC.

As a result, each of the lipases was conjugated to hydroxyapatite at 0.31, 0.26 and 0.48 mg/g, respectively. This result is a small amount compared to EGFP. This result is considered to be because the hydrophobic surface of lipase is difficult to interact with the surface of hydroxyapatite because the surface of hydroxyapatite is very polar.

Example 2: Preparation of hydroxyapatite using lipase-conjugated-hydroxyapatite bound by a linker

As the linker compound, 6-aminohexanoic acid (6-AmHA) was selected. The amino group of 6-aminohexanoic acid (6-AmHA) can be used for conjugation on the hydroxyapatite surface, and the carboxylate group can be linked to lipase. In addition, since the linker has 5 carbons between the amino group and the carboxylate group, it can help to reduce the repulsive action between the surface of hydroxyapatite and lipase. In addition, the linker is conjugated with hydroxyapatite after activation by a peptide coupling reagent.It is relatively inexpensive as a coupling reagent, and by-products (N,N'-diisopropyl urea) can be conveniently removed by washing with an organic solvent. A possible DIC was used. The specific method is as follows.

Hydroxyapatite (500 mg) was activated with DIC (N,N'-diisopropylcarbodiimide; 500 μl in 5 mL of dichloromethane) for 1 hour at 25°C. After washing three times with dichloromethane (1 mL), the activated hydroxyapatite was suspended in a solution of dichloromethane (5 mL) containing 6-aminohexane acid (500 mg). The suspension was incubated at 25° C. for 24 hours. After centrifuging the suspension, the precipitate was successively washed with dichloromethane, acetone, and water (1 mL for each solvent). Then, a solution of EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; 20 mg) in MES buffer (10 mL, pH 5.0, 100 mM) was mixed with the precipitate. The mixture was reacted at 25° C. for 20 minutes, and the precipitate was washed 3 times with BES buffer (1 mL, pH 7.2, 5 mM). The precipitate was resuspended in a lipase, Burkholderia cepacia lipase (BCL) or CRL (Candida rugosa lipase) solution (1 mL, 1.5 mg/mL in BES buffer), and the mixture was incubated for 24 hours. The lipase-conjugated hydroxyapatite was recovered by centrifugation and then dried at room temperature.

Experimental Example 3: Confirmation of conjugation of lipase-conjugation-hydroxyapatite

The conjugation of the linker in the lipase-conjugated-hydroxyapatite prepared in Example 2 was confirmed by NMR analysis after the decomposition step with a _{deuterium chloride (DCl) solution (20% in D 2 O), and the result} Is shown in FIG. 3.

As a result, as can be seen in FIG. 3, characteristic peaks of the linker compound can be confirmed in the NMR spectrum of the decomposed sample of linker-conjugated hydroxyapatite (6-AmHA-HAp). These results show that the linker compound is covalently bonded on the hydroxyapatite.

Experimental Example 4: Lipase-conjugated-hydroxyapatite properties confirmation

6-AmHA-HAp of Example 2 was activated with DCC, DIC or EDC. Then, CRL, which is a lipase, was conjugated to hydroxyapatite conjugated with an activated linker. The amount of bound CRL was determined to be 1.7, 2.5 and 2.8 mg/g, respectively.

Then, the reaction conversion rate in the transesterification reaction of (±)-1-phenylethyl alcohol and vinyl butyrate was compared, and the results are shown in FIG. 4.

As a result, as can be seen in Fig. 4, the one conjugated by EDC activation showed the highest conversion rate after 24 hours (Fig. 4). Thus, it can be seen that EDC is most suitable for binding BCL and CAL-B to linker-attached hydroxyapatite.

In addition, the state of the conjugated hydroxyapatite was confirmed through analysis of scanning electron microscopy (SEM) and powder X-ray diffraction (PXRD). FE-SEM images were obtained by JSM-7500F (Jeol, Japan), and XRD patterns were analyzed using a Bruker D8 Focus with a Cu target and X-ray tube operating at 40 kV and 40 mA. The results are shown in FIGS. 5 and 6.

As a result, as can be seen in Figures 5 and 6, it can be seen that the conjugation process does not change the morphology and crystallinity of the hydroxyapatite to which the linker and lipase are bound.

In addition, through IR analysis, the peak of the hydroxyapatite to which the linker and lipase were bound was confirmed, and the results are shown in FIG. 7.

As a result, as can be seen in FIG. 7, it can be seen that all characteristic peaks of the infrared spectroscopy analysis are preserved.

Test Example 5: Comparison of inactive form and inactive form of free form of hydroxyapatite conjugated with lipase

The specific activity of lipase-conjugated-hydroxyapatite (HAp-6-AmHA) on the transesterification reaction of vinyl butyrate and (±)-1-phenylethyl alcohol was compared with the free forms of the three lipases. It is shown in Table 2.

enzyme Specific activity value (μmol·min1 ^-One Mg ^-One ) Optical isomer ratio ( E ) One HAp(hydroxyapatite) n.d. n.d. 2 HAp-6-AmHA n.d. n.d. 3 Free CAL-B 0.070 ± 0.013 >200 4 HAp-6-AmHA-CAL-B 2.1 ± 0.082 >200 5 Free BCL 0.20 ± 0.0060 >200 6 HAp-6-AmHA-BCL 0.54 ± 0.12 >200 7 Free CRL 0.14 ± 0.0074 >200 8 HAp-6-AmHA-CRL 0.15 ± 0.014 >200

As a result, as can be seen in yields 1 and 2 of Table 2, the unmodified hydroxyapatite or the hydroxyapatite to which a linker is attached alone did not show any catalytic activity. In contrast, the three lipases conjugated to hydroxyapatite showed up to 30 times higher activity than the free enzyme. Since immobilization can separate the enzyme molecules from each other, the active site of the enzyme is more exposed to the substrate, and the activity increases compared to the free form of the enzyme. These results mean that the conjugation process of the present invention does not inactivate the lipase and does not change the structural characteristics of the lipase.

Experimental Example 6: Recycling experiment of lipase conjugated to hydroxyapatite

One of the important advantages of biocatalyst immobilization is that the immobilized biocatalyst can be recycled to reduce economic costs. Therefore, a recycling experiment of hydroxyapatite to which three lipases were bound was conducted.

Specifically, (±)-1-phenylethanol (0.05) in diisopropyl ether (1 mL) containing 50 mg of lipase-conjugated-hydroxyapatite (CRL and BCL), 5 mg of CAL-B). mmol) and vinyl butyrate (0.15 mmol) were added and reacted. The reaction was incubated at 25° C. for 24 hours. After the first reaction was completed, the lipase-conjugated-hydroxyapatite was washed three times with diisopropyl ether (1 mL). Then, the washed lipase-conjugated-hydroxyapatite was added to a solution of (±)-1-phenyl ethanol (0.05 mmol) and vinyl butyrate (0.15 mmol) in diisopropylether (1 mL). This step was repeated 9 times. Conversion at each step was analyzed by GC. Gas chromatography was analyzed with an Agilent 6890N using a chiral capillary column (Cyclosil-B 30 m x 0.25 mm, Agilent Technologies, Seoul, Korea). The reaction conversion rates were compared for 24 hours, and the results are shown in FIG. 8.

As a result, as can be seen in FIG. 8, in the case of CRL-conjugated-hydroxyapatite, the conversion rate was slightly changed in each cycle, but activity of 85% or more was still maintained after recycling 10 times. On the other hand, the activity of BCL- and CAL-B conjugated-hydroxyapatite was maintained even after 10 times of recycling. These results suggest that hydroxyapatite lime can be effectively used as a support material in biocatalyst immobilization.

Claims (14)

By a peptide-coupling agent, the amino group of the following linker compound is covalently bonded to the following hydroxyapatite, and the carboxylate group of the following linker compound is covalently bonded to the following hydrophobic protein,
The following linker compound is 6-aminohexanoic acid (6-AmHA),
The peptide-coupling agent is one or more bioconjugated complexes selected from the group consisting of N,N'-diisopropylcarbodiimide (DIC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC):
(i) hydroxyapatite;
(ii) a linker compound containing an amino group and a carboxylate group; And
(iii) hydrophobic protein.
delete delete The method of claim 1,
The hydrophobic protein is a bioconjugation complex, characterized in that the lipase.
The method of claim 4,
The lipase is one or more lipases selected from the group consisting of Candida lugosa lipase (CRL), Burkholderia sepacia lipase (BCL), and Candida antarctica lipase B (CAL-B). .
A first step of mixing and activating hydroxyapatite with a peptide-coupling agent;
A second step of suspending the activated hydroxyapatite in the first step in a linker compound solution;
A third step of centrifuging the suspension of the second step to obtain a precipitate, and then washing the precipitate;
A fourth step of mixing and culturing the precipitate of the third step with a peptide-coupling agent;
A fifth step of resuspending the precipitate of the fourth step in a solution containing a hydrophobic protein and then culturing; And
A sixth step of centrifuging the solution of the fifth step to obtain a complex to which a hydrophobic protein is bound; includes,
The peptide-coupling agent of the first and fourth steps is at least one selected from the group consisting of N,N'-diisopropylcarbodiimide (DIC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),
The linker compound of the second step is 6-aminohexanoic acid (6-AmHA) containing an amino group and a carboxylate group,
In the fourth step, the amino group of the linker compound is covalently bonded with the hydroxyapatite,
In the fifth step, the carboxyl group of the linker compound is covalently bonded to the hydrophobic protein.
delete delete delete The method of claim 6,
The method of manufacturing a bioconjugation complex, characterized in that the hydrophobic protein of the fifth step is a lipase.
A tissue engineering scaffold comprising the bioconjugation complex of claim 1 or the bioconjugation complex prepared by the manufacturing method of claim 6.
A medical material for bone filling or dental cavity filling, including the bioconjugation complex of claim 1 or the bioconjugation complex prepared by the manufacturing method of claim 6.
A membrane for inducing tissue regeneration or shielding, comprising the bioconjugation complex of claim 1 or the bioconjugation complex prepared by the manufacturing method of claim 6.
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