A nano-protease based on a bimetallic two-dimensional organic nanoskeleton design

Abstract

This invention develops a nanoprotease based on a bimetallic two-dimensional organic nanoframework and explores its catalytic mechanism. This two-dimensional structure achieves a 3-5 fold increase in hydrolysis efficiency by maximizing the accessibility of active sites and reducing mass transfer resistance, significantly outperforming existing reports. Metal doping and organic ligand properties further enhance Lewis acidity, thereby optimizing peptide bond hydrolysis performance. The organic ligand induces protein unfolding through a hydrophobic interface, exposing protein cleavage sites and accelerating the enzymatic reaction rate. CeCuBDC exhibits excellent stability and superior reusability during protein hydrolysis. Furthermore, this material can hydrolyze various proteins and mixed protein samples, and demonstrates outstanding catalytic activity in selectively cleaving peptide bonds containing hydrophobic residues. As a two-dimensional bimetallic MOF nanoprotease, CeCuBDC shows broad prospects for proteomics applications.

Description

Technical Field

[0001] This invention belongs to the field of developing novel nanoproteases, specifically involving the efficient hydrolysis of proteins by constructing bimetallic two-dimensional MOF materials as novel nanoproteases. Background Technology

[0002] In proteomics and biotechnology, peptide bond hydrolysis plays a crucial role in protein structure determination. Currently, trypsin is the most commonly used natural protease for protein hydrolysis. However, its practical application is limited by poor stability, reusability, and significant interference during the analytical process. These limitations hinder the further development of proteomics research. Developing novel nanoproteases for protein hydrolysis has proven to be an effective strategy to address these problems. Nanoproteases are more stable and economical than natural enzymes and are considered effective alternatives. Currently, nanoproteases utilize Lewis metals to polarize peptide bonds, promoting protein hydrolysis. Inspired by this process, high-Lewis materials are synthesized for efficient protein hydrolysis.

[0003] Materials such as metal-organic clusters (MOCs), polyoxymetalates (POMs), and metal-organic frameworks (MOFs) have been developed as nanoproteases for peptide bond hydrolysis, primarily utilizing high Lewis content to hydrolyze proteins. However, despite their extensive development, these nanoproteases still face numerous limitations. For example, zirconium-based MOFs (such as MOF-808 and MIP-201) suffer from low hydrolysis efficiency due to their micropore size restricting the diffusion of macromolecular substrates, leading to increased mass transfer resistance, reduced active site utilization, and consequently, lower biocompatibility. In contrast, POMs are nanoscale metal-oxygen clusters formed by the high oxidation state of transition metal ions and oxygen. Although POMs have been used for protein hydrolysis, their homogeneous nature makes them difficult to separate and limits their reusability. Therefore, developing novel nanoproteases that combine high active site utilization, excellent reusability, and significant stability is both crucial and highly challenging.

[0004] Two-dimensional metal-organic frameworks (2D MOFs) are two-dimensional materials composed of bridging metal centers and organic ligands. Their unique structure exposes active sites, reducing mass transfer resistance between proteins and active sites, thereby improving the utilization efficiency of active sites. The excellent framework structure ensures good reproducibility and stability during hydrolysis. Current MOF nanoproteases use metal centers (such as Zr, Cu, Ce) as Lewis acids to polarize the carbonyl carbon in the peptide bond, making the peptide bond susceptible to nucleophilic attack, thus mimicking the function of natural proteases. However, low Lewis acidity in MOF nanoproteases results in low catalytic activity, leading to insufficient peptide bond polarization efficiency. Bimetallic doping can enhance Lewis acidity, becoming an effective strategy for improving the catalytic activity of nanoproteases. Therefore, nanoproteases designed based on bimetallic 2D metal-organic frameworks are expected to exhibit superior catalytic efficiency, excellent recyclability, and stability compared to existing nanoproteases. However, this promising design strategy has not yet been applied in the field of nanoproteases. Summary of the Invention

[0005] The purpose of this invention is to successfully construct a two-dimensional bimetallic metal-organic framework (CeCuBDC) through rational design to address the challenge of developing highly efficient and stable peptide bond hydrolysis nanoproteases. The CeCuBDC synthesized in this invention features a two-dimensional structure that enhances the accessibility of active sites, significantly reduces mass transfer resistance, and the synergistic effect of the bimetallic system enhances Lewis acidity, thereby significantly improving peptide bond hydrolysis efficiency. The CeCuBDC synthesized in this invention exhibits excellent hydrolysis efficiency, as well as excellent stability and reusability. This invention uses cytochrome C (CYC) as a model protein to demonstrate the superior performance of this novel nanoprotease.

[0006] The method first involves fully dissolving cerium ammonium nitrate ((NH4)2Ce(NO3)6) and copper nitrate trihydrate (Cu(NO3)2·3H2O) in N,N-dimethylformamide (DMF), then fully dissolving the organic ligand terephthalic acid (H2BDC) in DMF, and finally mixing the two together and synthesizing them by a solvothermal method.

[0007] To achieve the above objectives, the present invention is implemented according to the following technical solution:

[0008] A method for preparing a two-dimensional bimetallic MOF (CeCuBDC), characterized by comprising the following steps:

[0009] (1) Weigh 1.096 g of (NH4)2Ce(NO3)6 and 0.242 g of Cu(NO3)2·3H2O and dissolve them completely in 40 mL of DMF under magnetic stirring at 1000 rpm; weigh 0.664 g of H2BDC and dissolve them completely in 40 mL of DMF under magnetic stirring at 1000 rpm.

[0010] (2) Further, the solution obtained in step (1) above was mixed with water (20 mL) in a polytetrafluoroethylene-lined stainless steel autoclave. Solvent-thermal synthesis was carried out at 80 °C for 24 hours. The resulting blue precipitate CeCuBDC was washed three times with DMF and then three times with ethanol, and dried under vacuum at 65 °C for 6 hours.

[0011] (3) Further, the prepared two-dimensional bimetallic MOF material CeCuBDC has a rhomboid sheet morphology and the size of CeCuBDC is 10-15 μm.

[0012] (4) Furthermore, the two-dimensional bimetallic MOF material CeCuBDC fully exposes the active sites, reduces the mass transfer resistance, and can fully contact the protein. Its hydrolysis efficiency is significantly improved compared with traditional three-dimensional nanoprotease.

[0013] (5) Furthermore, the CeCuBDC not only has excellent hydrolysis efficiency in individual model proteins, but also exhibits high-efficiency hydrolysis activity in mixed protein systems.

[0014] (6) Furthermore, the CeCuBDC selectively cleaves peptide bonds containing hydrophobic segments during protein hydrolysis, exhibiting good selectivity, indicating its broad application prospects in proteomics.

[0015] (7) Furthermore, the CeCuBDC exhibits excellent stability and reusability, indicating its good practicality in proteomics research.

[0016] The advantages of this invention are:

[0017] (1) Innovative use of two-dimensional bimetallic MOF as nanoprotease, using two-dimensional materials to reduce mass transfer resistance, fully expose active sites, improve protein hydrolysis efficiency, and provide a novel nanoprotease construction strategy.

[0018] (2) The doping of bimetals and the role of organic ligands further enhance the Lewis properties of the material, thereby improving the hydrolysis activity. Organic ligands can induce protein unfolding through hydrophobic interfaces, which is beneficial to the exposure of enzyme cleavage sites and accelerates the hydrolysis efficiency.

[0019] (3) The present invention selectively cleaves peptide bonds containing hydrophobic residues during protein hydrolysis, demonstrating the potential application prospects of CeCuBDC in proteomics.

[0020] (4) This invention exhibits excellent stability and reusability in protein hydrolysis, significantly reducing the application cost of natural enzymes. As a nano-protease, it overcomes the limitations of instability and non-reusability of natural enzymes.

[0021] (5) This invention can be widely applied to the hydrolysis of different types of proteins and mixed proteins, and is a novel nano-protease material. Detailed Implementation

[0022] The present invention will be described in detail below with reference to the embodiments, but this does not constitute a limitation on the present invention.

[0023] Example 1: Optimization and kinetic investigation of CYC hydrolysis reaction conditions by CeCuBDC.

[0024] (1) The effect of protein hydrolysis reaction is affected by temperature, amount of material, incubation time and pH. In order to achieve the best hydrolysis reaction efficiency, the present invention investigated the reaction conditions of the prepared nano-proteinase CeCuBDC hydrolyzing CYC.

[0025] (2) Based on the principle of single-variable control, this study first investigated the effect of material dosage on the hydrolysis reaction through a systematic experimental design. CYC was used as a model protein, and CeCuBDC dosage gradients of 1.5, 3, 4.5, 7, 10, 16, and 21 mg / mL were designed. During the experiment, different amounts of CeCuBDC (70 µL) and CYC (1 mg / mL, 700 µL) were mixed, and the mixture was transferred to a 60 °C constant-temperature shaker for incubation for 16 h. After the reaction was complete, the supernatant was obtained by centrifugation at 10,000 rpm for 5 min. Subsequently, 2.5 µL of the supernatant was mixed with 2.5 µL of DHB matrix, dropped onto an MTP target plate, and air-dried. The activity was then detected using MALDI-TOF mass spectrometry; the optimal CeCuBDC dosage was found to be 10 mg / mL.

[0026] (3) Further, the incubation time was further investigated under the optimal material dosage obtained in (2). During the experiment, CeCuBDC (10 mg / mL, 70 µL) and CYC (1 mg / mL, 700 µL) were mixed, and the mixture was transferred to a 60 °C constant temperature shaker for different incubation times (4, 8, 12, 16, 18, 24 h). After the reaction was completed, the supernatant was obtained by centrifugation at 10,000 rpm for 5 min. Subsequently, 2.5 µL of the supernatant was mixed with 2.5 µL of DHB matrix, dropped onto an MTP target plate, and air-dried. The activity was then detected by MALDI-TOF mass spectrometry. With the increase of incubation time, the hydrolysis reaction effect improved, and the hydrolysis efficiency reached 86% at 16 h.

[0027] (4) Further, under the optimal material dosage and reaction time conditions obtained in (2) and (3), pH was further investigated. CYC (1 mg / mL) was dissolved in HEPES buffer at different pH values ​​(5.5, 6.5, 7.4, 8.0, 9.0, 10.0). During the experiment, CeCuBDC (10 mg / mL, 70 µL) and CYC (1 mg / mL, 700 µL) were mixed, and the mixture was transferred to a 60 °C constant temperature shaker for incubation for 16 h. After the reaction was completed, the supernatant was obtained by centrifugation at 10,000 rpm for 5 min. Subsequently, 2.5 µL of the supernatant was mixed with 2.5 µL of DHB matrix, dropped onto an MTP target plate, and air-dried. The activity was then detected by MALDI-TOF mass spectrometry. The hydrolysis effect improved with increasing pH, and the hydrolysis efficiency reached 86% at pH 8.0.

[0028] (5) Based on the optimal material dosage, reaction time, and pH conditions in (2) to (4), the incubation temperature was further investigated. During the experiment, CeCuBDC (10 mg / mL, 70 µL) and CYC (1 mg / mL, 700 µL) were mixed, and the mixture was transferred to shakers at different temperatures (20, 30, 40, 50, 60, 70 °C) for incubation for 16 h. After the reaction was complete, the supernatant was obtained by centrifugation at 10000 rpm for 5 min. Subsequently, 2.5 µL of the supernatant was mixed with 2.5 µL of DHB matrix, dropped onto an MTP target plate, and air-dried. The activity was then detected using MALDI-TOF mass spectrometry; the results showed that the optimal incubation temperature was 60 °C.

[0029] (6) Kinetic studies were conducted on CeCuBDC under optimal hydrolysis conditions. The kinetic parameters of this invention were investigated using the Michaelis-Menten equation. CYC was used as a model protein to measure the kinetic parameters of the material. The results showed that the Michaelis constant (K) of this invention... m The concentration was 8.01 mg / mL, and the maximum reaction rate V to CYC was... max The concentration was 0.53 mg / mL / h, indicating that the CeCuBDC of the present invention has excellent substrate affinity and catalytic efficiency.

[0030] Example 2: Investigation into the mechanism of CeCuBDC hydrolyzing protein.

[0031] (1) Synthesizing different bimetallic materials: ZrCuBDC, HfCuBDC, CeZrBDC and CeHfBDC, and synthesizing single metal materials: CeBDC and CuBDC, compared with CeCuBDC of the present invention.

[0032] (2) Comparative materials ZrCuBDC, HfCuBDC, CeZrBDC, and CeHfBDC were synthesized according to the CeCuBDC method: (NH4)2Ce(NO3)6 (1.096 g) and ZrO(NO3)2 (0.462 g) were dissolved in DMF (40 ml) and stirred at 1000 rpm for 2 hours. H2BDC (0.664 g) was dissolved in DMF (40 ml) and stirred at 1000 rpm for 2 hours. Subsequently, the metal ions and organic ligands were mixed in a polytetrafluoroethylene-lined stainless steel autoclave, and water (20 ml) was added. The solvothermal synthesis reaction lasted for 24 hours. CeZrBDC was synthesized at 100 °C, and CeHfBDC, ZrCuBDC, and HfCuBDC were synthesized at 80 °C. The resulting precipitates were washed three times with DMF and then three times with ethanol, and dried under vacuum at 65 °C for 6 hours.

[0033] (3) Preparation of the comparative material CeBDC: (NH4)2Ce(NO3)6 (0.242 g) was dissolved in DMF (8 mL), and H2BDC (0.332 g) was dissolved in DMF (16 mL). The two solutions were mixed with water (4 mL) in a stainless steel autoclave lined with polytetrafluoroethylene and solvothermal synthesis was carried out at 100 °C for 24 hours. The resulting white precipitate CeBDC was washed three times with DMF and then three times with ethanol. After vacuum drying at 60 °C for 12 hours, CeBDC was obtained.

[0034] (4) Preparation of the comparative material CuBDC: Cu(NO3)2·3H2O (0.242 g) and H2BDC (0.166 g) were dissolved in DMF (20 mL). The two solutions were mixed and transferred to a polytetrafluoroethylene-lined stainless steel autoclave, and reacted at 80 °C for 24 hours. The product was washed three times with DMF and then three times with ethanol, and dried under vacuum at 65 °C for 24 hours to obtain CuBDC.

[0035] (5) Based on the bimetallic comparative material prepared in (2), using CYC as the model protein, the hydrolysis activity of the prepared bimetallic material was compared with that of CeCuBDC to explore the role of metal.

[0036] (6) During the experiment, CeCuBDC (10 mg / mL, 70 µL) was mixed with CYC (1 mg / mL, 700 µL), ZrCuBDC (10 mg / mL, 70 µL) was mixed with CYC (1 mg / mL, 700 µL), HfCuBDC (10 mg / mL, 70 µL) was mixed with CYC (1 mg / mL, 700 µL), CeZrBDC (10 mg / mL, 70 µL) was mixed with CYC (1 mg / mL, 700 µL), and CeHfBDC (10 mg / mL, 70 µL) was mixed with CYC (1 mg / mL, 700 µL). The mixtures were then transferred to a 60 °C constant temperature shaker and incubated for 16 h. After the reaction was completed, the supernatant was obtained by centrifugation at 10,000 rpm for 5 min. Subsequently, 2.5 µL of the supernatant was mixed with 2.5 µL of DHB matrix, dropped onto an MTP target plate, and air-dried. The activity was then detected using MALDI-TOF mass spectrometry.

[0037] (7) When the metal Cu is determined, the hydrolysis efficiency of CeCuBDC prepared in this invention is compared with that of the comparative materials ZrCuBDC and HfCuBDC. It is found that the hydrolysis efficiency of CeCuBDC prepared in this invention for CYC reaches 86%, while the hydrolysis efficiency of HfCuBDC and ZrCuBDC, which are used as comparative materials, is only 23% and 36%, respectively. The hydrolysis efficiency of CeCuBDC for CYC is much higher than that of the other two materials, proving that Ce is the main hydrolysis site. When the metal Ce is determined, the hydrolysis efficiency of CeCuBDC prepared in this invention is compared with that of the comparative materials CeZrBDC and CeHfBDC. It is found that the hydrolysis efficiency of CeCuBDC prepared in this invention for CYC reaches 86%, while the hydrolysis efficiency of CeZrBDC and CeHfBDC, which are used as comparative materials, is only 54% and 45%, respectively. The hydrolysis efficiency of CeCuBDC for CYC is much higher than that of the other two materials, proving that Cu is the main hydrolysis site.

[0038] (8) In order to further explore the interaction between Ce and Cu, based on the metal materials prepared in (3) and (4), CYC was used as a model protein, and the prepared single metal materials were compared with CeCuBDC to explore the interaction between metals.

[0039] (9) During the experiment, CuBDC (10 mg / mL, 70 µL) was mixed with CYC (1 mg / mL, 700 µL), CeBDC (10 mg / mL, 70 µL) was mixed with CYC (1 mg / mL, 700 µL), and CeCuBDC (10 mg / mL, 70 µL) was mixed with CYC (1 mg / mL, 700 µL). The mixtures were then transferred to a 60 °C constant temperature shaker and incubated for 16 h. After the reaction was completed, the supernatant was obtained by centrifugation at 10,000 rpm for 5 min. Subsequently, 2.5 µL of the supernatant was mixed with 2.5 µL of DHB matrix, dropped onto an MTP target plate, and air-dried. The activity was then detected by MALDI-TOF mass spectrometry.

[0040] (10) The hydrolysis efficiency of CeCuBDC prepared by the present invention reaches 86% for CYC, while the hydrolysis efficiencies of CeBDC and CuBDC, which are used as comparative materials, are only 76% and 70%, respectively. The hydrolysis efficiency of CeCuBDC is much higher than that of the two single metal materials, indicating that Cu and Ce have a synergistic effect.

[0041] Example 3: Stability and reusability test of CeCuBDC.

[0042] (1) Thermal stability assessment: The thermal stability of CeCuBDC and natural trypsin (enzyme concentration of 0.5 mg / mL) was assessed by measuring their residual activity after incubation at 60°C for 240 min and cooling to room temperature. The activity test procedure was as follows: 700 µL LCYC (1 mg / mL) was mixed with 70 µL CeCuBDC (10 mg / mL) or the same amount of natural trypsin solution. The mixture was transferred to a 60°C constant temperature shaker and incubated for 16 hours. The mixture was then centrifuged at 10,000 rpm for 5 min to separate and obtain the supernatant. Subsequently, 2.5 µL of the supernatant was mixed with 2.5 µL of DHB matrix, dropped onto an MTP target plate, and air-dried. The activity was then detected using MADLDI-TOF. The hydrolytic activity of natural trypsin was only 12%, while CeCuBDC still maintained 83% of its activity, indicating that the prepared nanoproteinase has good thermal stability.

[0043] (2) Storage stability study: The storage stability of CeCuBDC and natural trypsin (enzyme concentration of 0.5 mg / mL) was evaluated by measuring their activities after 9 days of storage at room temperature. The activity test procedure was as follows: 700 µL of CYC (1 mg / mL) was mixed with 70 µL of CeCuBDC (10 mg / mL) or the same amount of natural trypsin solution. The mixture was transferred to a 60 °C constant temperature shaker and incubated for 16 hours. The mixture was then centrifuged at 10,000 rpm for 5 min to separate the supernatant. Subsequently, 2.5 µL of the supernatant was mixed with 2.5 µL of DHB matrix, dropped onto an MTP target plate, and air-dried. The activity was then detected using MADLDI-TOF. The results showed that only 30% of the activity of natural trypsin was retained, while CeCuBDC maintained high activity, demonstrating that CeCuBDC has good stability as a nano-protease.

[0044] (3) Reusability test: After each catalytic reaction, CeCuBDC was washed with deionized water and separated from the reaction mixture by centrifugation (10,000 rpm, 5 min) for reuse. 700 µL of CYC (1 mg / mL) was mixed with the material, and the mixture was transferred to a 60 °C constant temperature shaker for 16 hours. The supernatant was obtained by centrifugation at 10,000 rpm for 5 min. Subsequently, 2.5 µL of the supernatant was mixed with 2.5 µL of DHB matrix, dropped onto an MTP target plate, and air-dried. The activity was then detected using MADLDI-TOF. After 6 cycles, CeCuBDC still retained 72% of the catalytic activity, demonstrating that CeCuBDC has good reusability as a nano-proteinase.

[0045] Example 4: Detection of the hydrolytic activity of CeCuBDC on mixed proteins.

[0046] (1) Bovine serum albumin (BSA), equine myoglobin (Mb), egg white lysozyme (HEWL) and CYC were selected as model proteins. The proteins were mixed in pairs to prepare a binary mixed protein solution for subsequent hydrolysis activity detection.

[0047] (2) Preparation of mixed proteins: First, prepare solutions of different binary mixed proteins, prepare a binary mixed solution containing CYC and BSA (enzyme concentration of 1 mg / mL), prepare a binary mixed solution containing Mb and BSA (enzyme concentration of 1 mg / mL), and prepare a binary mixed solution containing HEWL and BSA (enzyme concentration of 1 mg / mL).

[0048] (3) Evaluation of the hydrolysis efficiency of the mixed protein: In the experiment, CeCuBDC (10 mg / mL, 70 µL) was mixed with a binary mixed protein (1 mg / mL, 700 µL), and the mixture was transferred to a 60 °C constant temperature shaker for 16 h. After the reaction was completed, the supernatant was obtained by centrifugation at 10,000 rpm for 5 min. Then, 2.5 µL of the supernatant was mixed with 2.5 µL of DHB matrix, dropped onto an MTP target plate, and air-dried. The activity was then detected by MALDI-TOF mass spectrometry.

[0049] (4) Further, the results showed that in the BSA-CYC mixed system, the hydrolysis efficiency of BSA reached 84%, while that of CYC was 76%. In the BSA-Mb mixed system, the hydrolysis efficiency of BSA reached 84%, while that of Mb reached 99%. In the BSA-HEWL mixed system, the hydrolysis efficiency of BSA was 83%, while that of HEWL was 98%. The results indicate that CeCuBDC exhibits high hydrolytic activity for complex protein mixtures.

Claims

1. A method for preparing nanoprotease based on a bimetallic two-dimensional organic nanoframework, characterized in that, Includes the following steps: (1) Weigh 1.096 g of (NH4)2Ce(NO3)6 and 0.242 g of Cu(NO3)2·3H2O and dissolve them completely in 40 mL of DMF under magnetic stirring at 1000 rpm; Weigh 0.664 g of H2BDC and dissolve them completely in 40 mL of DMF under magnetic stirring at 1000 rpm; (2) The solution obtained in the above steps was mixed with water (20 mL) in a polytetrafluoroethylene-lined stainless steel autoclave; the solvothermal synthesis was carried out at 80°C for 24 hours; the resulting blue precipitate CeCuBDC was washed multiple times with DMF and ethanol and dried under vacuum at 65°C for 6 hours.

2. A nanoprotease based on a bimetallic two-dimensional organic nanoframework, prepared according to the method of claim 1.