Core-satellite-shaped metal organic framework connected by DNA and preparation method and application of biological enzyme microreactor composite material of core-satellite-shaped metal organic framework

Abstract

The invention discloses a core-satellite-shaped metal organic framework based on DNA connection and a preparation method and application of a bio-enzyme microreactor composite material of the core-satellite-shaped metal organic framework. Bio-enzymes are fixed in different types of metal organic framework materials in an in-situ synthesis mode to form an enzyme (at) MOF composite material; and directionally connecting the large-size metal organic framework core unit with the small-size metal organic framework satellite unit by using a DNA bridging strategy to form a core-satellite-shaped metal organic framework biological enzyme microreactor composite material, and applying the composite material to catalytic reactions of different biological enzymes. According to the invention, an ordered self-assembly system constructed by DNA can accurately regulate and control the spatial arrangement of enzymes, a substrate transfer path and a reaction microenvironment, so that the efficient synergy of incompatible cascade enzymes is realized on the premise of maintaining the self activity of the enzymes, and the method is suitable for various complex multi-enzyme catalytic systems; the catalytic efficiency, the thermal stability, the solvent tolerance and the reusability of the system are improved.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering, specifically relating to the preparation method and application of a DNA-linked core-satellite metal-organic framework and its bio-enzyme microreactor composite material. Background Technology

[0002] In biological metabolic pathways, cascade enzyme synergistic catalysis is key to achieving efficient metabolism. Multi-enzyme cascade systems offer significant advantages over single-enzyme catalysis: they eliminate intermediate product separation steps, enable efficient reactant transfer, and allow for precise control of reaction rates, making them a reliable route for the green synthesis of fine chemicals. However, the poor stability of natural enzymes and the conflicting reaction conditions (such as pH / temperature) between different enzymes severely restrict the large-scale application of cascade reactions.

[0003] To address the limitations of free enzymes, existing strategies mainly include: directed enzyme evolution (e.g., the Arnold team) to improve stability; gene / protein engineering (e.g., the Dannert team) to enhance performance through amino acid substitution; and enzyme modification (e.g., the Craik team) to improve substrate recognition and catalytic ability. Furthermore, enzyme immobilization is a key strategy for improving enzyme activity, stability, and selectivity, laying the foundation for industrial applications.

[0004] Metal-organic frameworks (MOFs), with their unique structure, rigid conformation, and customizable pores, are ideal carriers for multi-enzyme immobilization, protecting enzymes and promoting selective substrate diffusion. Chen et al. utilized MOF / COF complexes to provide enzymes with high conformational freedom and environmental adaptability; Li et al. developed multilayer encapsulated MOF structures for cascade enzymes. However, existing technologies in the field of cascade enzyme immobilization still have significant limitations: on the one hand, most enzyme immobilization strategies rely on harsh conditions such as high temperature, strong acids and bases, or organic reagents, which are difficult to match the mild environmental requirements of cascade enzymes, easily leading to conformational damage to enzyme activity; on the other hand, the lack of effective spatial compartmentalization structural design makes it impossible to precisely separate and load incompatible cascade enzymes with significantly different microenvironmental requirements, causing the two types of enzymes to interfere with each other in the same system, making it difficult to maintain independent function; at the same time, the existing immobilization carriers have insufficient microenvironmental regulation capabilities, failing to provide independent and adjustable local environments for each cascade enzyme, ultimately making it difficult to achieve microenvironmentally adjustable, precisely ordered, and customizable multilayer MOF enzyme reactors, severely limiting the improvement of cascade catalytic efficiency and adaptability to complex biocatalytic scenarios. Summary of the Invention

[0005] Objective: To address the problems of insufficient spatial separation, limited microenvironment regulation, and poor recovery stability in existing MOF multi-enzyme immobilization systems, this invention proposes for the first time a core-satellite metal-organic framework (MOF) and its bio-enzyme microreactor composite material linked by DNA. This invention innovatively utilizes DNA molecules to bridge large-size core MOFs and small-size satellite MOFs, constructing a customizable spatial structure through MOP coordination bonds between phosphate groups and metal nodes. This effectively overcomes existing technological bottlenecks: it gently immobilizes cascade enzymes in MOF units of different sizes, providing independent microenvironments for incompatible enzymes through spatial separation, avoiding mutual interference; it also achieves substrate channelization to reduce intermediate product inhibition, significantly improving catalytic efficiency and protecting and enhancing the enzyme's thermal stability, solvent tolerance, and reusability; simultaneously, it uses green, tunable, and biocompatible DNA for precise assembly, with mild assembly conditions, convenient recovery and reuse, easy scale-up production, and modular adaptation to dual-enzyme and multi-enzyme systems, biomimetically mimicking cellular compartmentalized metabolism, providing a universal and efficient solution for complex biocatalysis.

[0006] The present invention also provides a method for preparing and applying the aforementioned DNA-linked core-satellite metal-organic framework bioenzyme microreactor composite material.

[0007] The core of this invention—a satellite-shaped metal-organic framework bio-enzyme microreactor composite material—can be used in various enzyme catalytic systems. For example, it can be applied to the spatially separated cascade reaction of glucose oxidase (GOx) and horseradish peroxidase (HRP); the synergistic synthesis of glyconucleotide donors through a dual-natural enzyme cascade reaction of N-acetylglucosamine 1-kinase (NahK) and uridine transferase (GlmU); the cascade reaction of lipase (CRL) and cytochrome P450 enzyme (CYP450) from Candida albicans to generate hydroxylauric acid; and the detection of lactose content through a three-enzyme cascade of β-galactosidase (β-Gal), glucose oxidase (GOx), and horseradish peroxidase (HRP). This invention offers operability, continuity, stability, and ease of practical application, while also being customizable, significantly reducing the cost of industrial-grade biocatalysis.

[0008] Technical solution: To achieve the above objectives, the present invention provides a core-satellite metal-organic framework connected by DNA, characterized in that the core-satellite metal-organic framework comprises: a large-sized core MOF unit, a small-sized satellite MOF unit, and bridging DNA, wherein the core MOF and the satellite MOF are directionally connected by MOP coordination bonds formed between the phosphate groups of the DNA and the metal centers on the MOF, thereby forming a core-satellite metal-organic framework material.

[0009] The core MOF unit has a diameter of 3-20 μm, and the satellite MOF unit has a diameter of 0.05-1 μm. The assembly formed by the DNA-linked core MOF unit and satellite MOF unit has a core-satellite structure, with a single core MOF connecting multiple satellite MOF units. The bridging DNA has a length of 8-24 deoxyribonucleotides and is a single-stranded DNA composed of any one of adenine, guanine, cytosine, or thymine.

[0010] Preferably, the core MOF unit is supported by one of a zeolite-like imidazole ester framework material and a zeolite-like pyrimidine framework material, and the satellite MOF unit is supported by a metal-organic framework material (such as Ni(ICA)2) with controlled size, and the satellite MOF unit has a diameter smaller than that of the core MOF unit.

[0011] The present invention discloses a core-satellite metal-organic framework bioenzyme microreactor composite material connected by DNA. The composite material uses the core-satellite metal-organic framework as a carrier to encapsulate different enzymes in the core MOF unit and the satellite MOF unit respectively (multiple enzymes can be loaded into the same unit as needed) to form a core-satellite metal-organic framework bioenzyme microreactor composite material.

[0012] The enzymes include at least two of the following: β-galactosidase (β-Gal), glucose oxidase (GOx), horseradish peroxidase (HRP), N-acetylglucosamine 1-kinase (NahK), uridine transferase (GlmU), lipase (CRL) produced by Candida albicans, and cytochrome P450 enzyme (CYP450).

[0013] Preferably, the enzyme solutions are β-galactosidase solution (β-Gal), glucose oxidase solution (GOx), horseradish peroxidase solution (HRP), N-acetylglucosamine 1-kinase solution (NahK), uridine transferase solution (GlmU), lipase solution produced by Candida albicans (CRL), and cytochrome P450 enzyme solution (CYP450).

[0014] The method for preparing the DNA-linked core-satellite metal-organic framework bioenzyme microreactor composite material of the present invention is characterized by comprising the following steps:

[0015] (1) Synthesis of enzyme@MOF core unit: The reaction solution of enzyme@MOF composite catalytic material is obtained by mixing and stirring the ligand solution, enzyme solution and metal ion solution, and centrifuging to obtain enzyme@MOF core unit composite material.

[0016] (2) Synthesis of enzyme@MOF satellite units: The reaction solution of enzyme-metal-organic framework composite catalytic material is obtained by mixing and stirring the ligand solution, enzyme solution and metal ion solution, and centrifuging to obtain enzyme@MOF satellite unit composite material.

[0017] (3) DNA directed assembly: The core unit and satellite unit are placed in an acidic environment and bridging DNA is added. After self-assembly, a core-satellite metal-organic framework bioenzyme microreactor composite material is obtained.

[0018] As a preferred method, a core-satellite enzyme-metal-organic framework system is synthesized by using pre-synthesized enzyme@MOF core units and enzyme@MOF satellite units as raw materials and single-stranded DNA as a linker, and then assembling them under acidic conditions.

[0019] Preferably, the enzyme@MOF core unit is synthesized in aqueous phase by co-precipitation: a ligand solution (one of 2-methylimidazolium, 2-formaldehyde imidazolium, 2-hydroxypyrimidine and 2-hydroxy-5-fluoropyrimidine), an enzyme solution, and a metal ion solution (one of zinc nitrate, zinc acetate, and cobalt acetate) are mixed and stirred to obtain a reaction solution of the enzyme@MOF core unit composite catalytic material, and the composite material is obtained after centrifugation.

[0020] Preferably, the enzyme@MOF satellite unit is synthesized in aqueous phase by co-precipitation: a ligand solution (one of 2-methylimidazolium, 2-formaldehyde imidazolium, 2-hydroxypyrimidine and 2-hydroxy-5-fluoropyrimidine), an enzyme solution, and a metal ion solution (one of zinc nitrate, zinc acetate, nickel nitrate, and cobalt acetate) are mixed and stirred to obtain a reaction solution of enzyme@MOF composite catalytic material. After centrifugation, the enzyme@MOF satellite unit composite material is obtained.

[0021] In step (1), the ligand is one of 2-methylimidazole, 2-formaldehydeimidazole, 2-hydroxypyrimidine and 2-hydroxy-5-fluoropyrimidine, and the salt of the metal ion is one of zinc nitrate, zinc acetate and cobalt acetate; the molar ratio of ligand to metal ion is 1:4 to 8:1, the enzyme content in the ligand solution, enzyme solution and metal ion solution system is 1 to 4 mg / mL, the reaction temperature is 40 to 50℃, the stirring is 200 to 400 rpm, and the reaction time is 10 to 15 h.

[0022] Preferably, the centrifugation speed is 8000 rpm and the time is 6 min.

[0023] In step (2), the ligand is one of 2-methylimidazole, 2-formaldehydeimidazole, 2-hydroxypyrimidine and 2-hydroxy-5-fluoropyrimidine, and the metal ion salt is one of zinc nitrate, zinc acetate, nickel nitrate and cobalt acetate; the molar ratio of ligand to metal ion is 1:4 to 8:1, the enzyme content in the ligand solution, enzyme solution and metal ion solution system is 1 to 4 mg / mL, the reaction temperature is 40 to 50℃, the stirring is 200 to 400 rpm, and the reaction time is 0.5 to 2 h.

[0024] In step (3), the acidic environment is a Tris-HCl solution, citric acid solution, or PBS solution with a concentration of 0.2-2 mol / L and a pH of 4-6; the enzyme@MOF core unit composite material is 30-50 mg; the enzyme@MOF satellite unit is 60-100 mg; the DNA concentration is 10-15 mmol / L and the volume is 200-500 μL; the self-assembly time is 10-70 min and the temperature is room temperature.

[0025] The present invention relates to the application of the DNA-linked core-satellite metal-organic framework bioenzyme microreactor composite material in different multi-enzyme cascade catalytic systems.

[0026] The DNA-linked core-satellite metal-organic framework bio-enzyme microreactor composite material of the present invention uses enzyme@MOF core units and enzyme@MOF satellite units as raw materials and single-stranded DNA as a linker. It is synthesized into a core-satellite enzyme-metal-organic framework system by post-assembly under acidic conditions. The present invention is the first to use DNA in the synthesis process of core-satellite metal-organic framework bio-enzyme microreactor composite material.

[0027] The present invention relates to the application of the DNA-linked core-satellite metal-organic framework bioenzyme microreactor composite material in one or more cascade bioenzyme catalytic systems.

[0028] The applications include: detecting glucose content using a glucose oxidase (GOx) and horseradish peroxidase (HRP) cascade system to prepare a glucose detector; synergistically synthesizing glyconucleotide donors using a dual natural enzyme cascade system of N-acetylglucosamine 1-kinase (NahK) and uridine transferase (GlmU); generating hydroxylauric acid using a cascade system of lipase (CRL) and cytochrome P450 enzyme (CYP450) from Candida albicans; and detecting lactose content using a three-enzyme cascade system of β-galactosidase (β-Gal), glucose oxidase (GOx), and horseradish peroxidase (HRP). The enzyme activity test results were characterized using spectroscopy, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, nuclear magnetic resonance, liquid chromatography, and TLC, and the relative activities were calculated.

[0029] Furthermore, the application includes the following steps:

[0030] (1) The GOx&HRP core-satellite metal-organic framework bio-enzyme microreactor composite material (GOx&HRP@core-satellite MOF) was added to a mixed solution of glucose and tetramethylbenzidine (TMB) for glucose detection reaction. After the reaction was completed in a metal bath, the supernatant was collected by centrifugation and the absorbance was measured at 652 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The relative activity was then calculated.

[0031] (2) The NahK&GlmU core-satellite metal-organic framework bioenzyme microreactor composite material (NahK&GlmU@core-satellite MOF) was added to Tris-HCl buffer containing ATP, UTP, monosaccharide substrate GlcNAc (10 mmol / L), and MgCl2. After the reaction was completed in a metal bath, the supernatant was collected by centrifugation. The generated glyconucleotide UDP-GlcNAc was qualitatively and quantitatively detected by liquid chromatography, and its relative activity was calculated.

[0032] (3) The CRL&CYP450-core-satellite metal-organic framework bioenzyme microreactor composite material (CRL&CYP450@core-satellite MOF) was added to a mixed reaction solution of p-nitrophenyl laurate and H2O2 to generate hydroxylauric acid. After the reaction in the metal bath was completed, it was extracted with ethyl acetate under acidic conditions. The centrifugation speed was 8000 rpm and the time was 6 min. The content of hydroxylauric acid generated was qualitatively and quantitatively detected by gas chromatography-mass spectrometry and its relative activity was calculated.

[0033] (4) The β-Gal&GOx&HRP-core-satellite metal-organic framework bioenzyme microreactor composite material (β-Gal&GOx&HRP@core-satellite MOF) was added to a mixed solution of lactose and tetramethylbenzidine (TMB) to carry out the lactose detection reaction. After the reaction was completed in the metal bath, the supernatant was centrifuged and the absorbance was measured at 652 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The relative activity was then calculated.

[0034] In step (1), the mass of the GOx&HRP-core-satellite metal-organic framework bioenzyme microreactor composite material (GOx&HRP@core-satellite MOF) is 10% of the mass of the substrate glucose. The glucose reaction solution is 0.4 mL, 10 mmol / L, the metal bath temperature is 30~50℃, the reaction time is 10~30 min, the rotation speed is 600~800 rpm, the centrifugation speed is 8000 rpm, and the time is 6 min.

[0035] In step (2), the mass of the NahK&GlmU-core-satellite metal-organic framework bioenzyme microreactor composite material (NahK&GlmU@core-satellite MOF) is 10% of the substrate monosaccharide mass. The total ATP concentration in the reaction solution is 5-12 mmol / L, the UTP concentration is 5-15 mmol / L, the monosaccharide substrate GlcNAc concentration is 10-15 mmol / L, the solvent is 100 mmol / L Tris-HCl buffer at pH 7.5, the metal bath temperature is 20-40℃, the reaction time is 24-48 h, the rotation speed is 600-800 rpm, the centrifugation speed is 8000 rpm, and the time is 6 min. High performance liquid chromatography conditions: Inertsil ODS-3 column (150 mm × 4.6 mm, 5 μm). Mobile phase A is phosphate buffer composed of 18 mmol / L sodium dihydrogen phosphate and disodium hydrogen phosphate (pH adjusted to 6.8 with phosphate), and mobile phase B is pure methanol. The sample pan temperature and chromatographic temperature were both 25°C. The detection wavelength was 272 nm, and the injection volume was 20 μL. Mass spectrometry detection of glyconucleotides was performed in negative ion mode.

[0036] In step (3), the CRL&CYP450-core-satellite metal-organic framework bioenzyme microreactor composite material (CRL&CYP450@core-satellite MOF) is 10% of the substrate p-nitrophenyl laurate. The concentration of p-nitrophenyl laurate in the entire reaction solution is 1~3 mmol / L, the concentration of H2O2 is 20~30 mmol / L, the metal bath temperature is 20~40℃, the reaction time is 24~48 h, the rotation speed is 600~800 rpm, and after the reaction, it is extracted with ethyl acetate under acidic conditions, centrifuged at 8000 rpm for 6 min. The supernatant is taken and the content of hydroxylauric acid is detected by gas chromatography-mass spectrometry. Gas chromatography-mass spectrometry conditions: initial temperature is 100℃, held for 0.5 min, heating rate is 10℃ / min, and the temperature is increased to 300℃. Helium gas flows through the column at 0.7 mL / min, and the injection temperature is 250℃. Split ratio: 99:1. Ionization method: Electron impact ionization (70 eV). Mass scan range: m / z 33–400.

[0037] In step (4), the β-Gal&GOx&HRP-core-satellite metal-organic framework bioenzyme microreactor composite material (β-Gal&GOx&HRP@core-satellite MOF) is 10% of the substrate lactose mass. The lactose reaction solution is 0.4 mL, 10 mmol / L, the metal bath temperature is 30~50℃, the reaction time is 10~30 min, the rotation speed is 600~800 rpm, the centrifugation speed is 8000 rpm, and the time is 6 min.

[0038] Specifically, the preparation of the present invention preferably includes four parts:

[0039] Part 1: Preparation of Enzyme@MOF Core Units

[0040] A ligand solution, an enzyme solution, and a metal salt solution are mixed. The ligand solution is selected from one of 2-methylimidazole, 2-formaldehyde imidazole, 2-hydroxypyrimidine, and 2-hydroxy-5-fluoropyrimidine. The metal salt is selected from one of zinc nitrate, zinc acetate, and cobalt acetate. The mixture is stirred and reacted in an aqueous phase, then centrifuged. After mixing and stirring, the reaction solution of the enzyme@MOF core unit is obtained. After centrifugation, the enzyme@MOF core unit composite material is obtained. The molar ratio of ligand to metal ion is 1:4 to 8:1. The enzyme solutions are glucose oxidase solution & horseradish peroxidase solution (GOx & HRP), N-acetaminohexose 1-position kinase solution & uridine transferase solution (NahK & GlmU), lipase solution from Candida albicans & cytochrome P450 enzyme solution (CRL & CYP450), and β-galactosidase solution & glucose oxidase solution & horseradish peroxidase solution (β-Gal & GOx & HRP). The reaction temperature is 40~50℃, the stirring is done with a magnetic stirrer at a speed of 200~400 rpm, and the reaction time is 10~15 h.

[0041] Part 2: Preparation of enzyme@MOF satellite units

[0042] A ligand solution, an enzyme solution, and a metal salt solution are mixed. The ligand solution is selected from one of 2-methylimidazole, 2-formaldehyde imidazole, 2-hydroxypyrimidine, and 2-hydroxy-5-fluoropyrimidine. The metal salt is selected from one of zinc nitrate, zinc acetate, nickel nitrate, and cobalt acetate. The mixture is stirred and reacted in an aqueous phase, then centrifuged. After mixing and stirring, the reaction solution of the enzyme@MOF satellite unit is obtained. After centrifugation, the enzyme@MOF satellite unit composite material is obtained. The concentration ratio of the ligand solution to the metal salt solution is 1:4 to 8:1. The enzyme solutions are β-galactosidase solution (β-Gal), glucose oxidase solution (GOx), horseradish peroxidase solution (HRP), N-acetaminohexose 1-kinase solution (NahK), uridine transferase solution (GlmU), lipase solution produced by Candida albicans (CRL), and cytochrome P450 enzyme (CYP450). The reaction temperature is 40~50℃, the stirring is done with a magnetic stirrer at a speed of 200~400 rpm, and the reaction time is 10~15h.

[0043] Part 3: Synthesis of a DNA-linked core-satellite metal-organic framework bio-enzyme microreactor composite material through post-processing. The enzyme@MOF core unit obtained in step (1) and the enzyme@MOF satellite unit obtained in step (2) were placed in an acidic environment, and single-stranded DNA was added. The DNA phosphate groups were directionally self-assembled through the MOP coordination bonds between the MOF metal center to form a core-satellite metal-organic framework bio-enzyme microreactor composite material with a spatially separated enzyme system. The acidic environment was a Tris-HCl solution, citric acid solution, or PBS solution with a concentration of 0.2~2 mol / L and a pH of 4~6; the amount of core MOF unit was 30~50 mg; the amount of satellite MOF unit was 60~100 mg; the length of single-stranded DNA was 8~24 deoxyribonucleotides, the bases were adenine, guanine, cytosine, and thymine, the concentration was 10~15 mmol / L, and the added volume was 200~500 μL; the self-assembly time was 10~70 min, and the temperature was room temperature.

[0044] Part Four: Practical Applications of Core-Satellite Metal-Organic Framework Bioenzyme Microreactor Composites

[0045] The core-satellite metal-organic framework bioenzyme microreactor composite material, with a mass of 10% of the substrate mass, was used to detect glucose content in a cascade reaction of glucose oxidase (GOx) and horseradish peroxidase (HRP); a dual natural enzyme cascade reaction of N-acetylglucosamine 1-kinase (NahK) and uridine transferase (GlmU) synergistically synthesized glyconucleotide donors; a cascade reaction of lipase (CRL) and cytochrome P450 enzyme (CYP450) produced from Candida albicans generated hydroxylauric acid; and a three-enzyme cascade of β-galactosidase (β-Gal), glucose oxidase (GOx), and horseradish peroxidase (HRP) was used to detect lactose content.

[0046] (1) 10% by weight of the substrate GOx&HRP-core-satellite metal-organic framework bioenzyme microreactor composite material (GOx&HRP@core-satellite MOF) was added to 0.4 mL of 10 mmol / L glucose reaction solution for glucose detection. The reaction was carried out in a metal bath at a temperature of 30-50℃ for 10-30 min at a rotation speed of 600-800 rpm. After the reaction was completed, the supernatant was collected by centrifugation at 8000 rpm for 6 min. The absorbance was measured at 652 nm using an enzyme-linked immunosorbent assay (ELISA) reader to calculate the relative activity.

[0047] (2) 10% (by mass) of the NahK & GlmU core-satellite metal-organic framework bioenzyme microreactor composite material (NahK & GlmU@core-satellite MOF) was added to 100 mmol / L Tris-HCl buffer at pH 7.5, containing ATP (10 mmol / L), UTP (10 mmol / L), monosaccharide substrate GlcNAc (10 mmol / L), and MgCl2 (2 mmol / L). The reaction was carried out at 37°C for 24 h. The glycosyl donor (uridine diphosphate-N-acetylglucosamine) was detected by high performance liquid chromatography (HPLC) and mass spectrometry (MS / MS). HPLC conditions: Inertsil ODS-3 column (150 mm × 4.6 mm, 5 μm). Mobile phase A was phosphate buffer consisting of 18 mmol / L sodium dihydrogen phosphate and disodium hydrogen phosphate (pH adjusted to 6.8 with phosphate), and mobile phase B was pure methanol. The sample pan temperature was 25°C, and the chromatographic temperature was 25°C. The detection wavelength was 272 nm, and the injection volume was 20 μL. Mass spectrometry was used to detect glyconucleotides in negative ion mode.

[0048] (3) The CRL&CYP450-core-satellite metal-organic framework bioenzyme microreactor composite material (CRL&CYP450@core-satellite MOF) at 10% of the substrate mass was added to 1.5 mL of 2 mmol / L p-nitrophenyl laurate reaction solution for hydrolysis and hydroxylation. The H2O2 concentration was 30 mmol / L, the metal bath temperature was 20~40℃, the reaction time was 24~48 h, and the rotation speed was 600~800 rpm. After the reaction, the mixture was extracted with ethyl acetate under acidic conditions, centrifuged at 8000 rpm for 6 min. The supernatant was collected and detected by gas chromatography-mass spectrometry (GC-MS). GC-MS conditions: initial temperature 100℃, held for 0.5 min, heating rate 10℃ / min, increased to 300℃. Helium gas flowed through the column at 0.7 mL / min, and the injection temperature was 250℃. Split ratio: 99:1. Ionization method: Electron impact ionization (70 eV). Mass scan range: m / z 33–400.

[0049] (4) 10% by weight of the β-Gal&GOx&HRP-core-satellite metal-organic framework bioenzyme microreactor composite material (β-Gal&GOx&HRP@core-satellite MOF) was added to 0.4 mL of 10 mmol / L lactose reaction solution for lactose detection. The reaction was carried out in a metal bath at a temperature of 30-50℃ for 10-30 min at a rotation speed of 600-800 rpm. After the reaction was completed, the supernatant was collected by centrifugation at 8000 rpm for 6 min. The absorbance was measured at 652 nm using an enzyme-linked immunosorbent assay (ELISA) reader to calculate the relative activity.

[0050] By employing the aforementioned combined process, enzymes are immobilized within a metal-organic framework (MOF), reducing the impact of the environment on the enzymes, enhancing their mechanical properties, and improving operational stability. Simultaneously, through the coordination of DNA phosphate groups with the MOPs of the MOF metal centers, large-sized enzyme@MOF core units and small-sized enzyme@MOF satellite units are directionally assembled into a core-satellite hierarchical structure. This creates an independently adjustable microenvironment for enzyme cascade reactions, enabling not only highly efficient synergistic reactions of incompatible cascade enzymes but also flexible configuration of dual-enzyme and multi-enzyme combinations, suitable for various scenarios such as cascade catalysis and biosynthesis. Spatial separation facilitates substrate channelization, mitigating intermediate inhibition. The core-satellite MOF bioreactor composite material constructed in this invention can efficiently exert the synergistic catalytic activity of multiple enzymes; it inherits the advantages of MOF materials such as large specific surface area and chemical stability; it improves enzyme catalytic efficiency and product conversion rate; and it provides a universal platform for constructing cell-inspired programmable multi-compartment bioreactors. This system possesses customizability, high efficiency, and practical application value.

[0051] This invention represents a groundbreaking innovation: utilizing DNA to connect large-sized core MOFs (3–20 μm) with small-sized satellite MOFs (0.05–1 μm) to construct highly ordered core-satellite assemblies. Its breakthroughs lie in: independently customizable microenvironments: the core / satellite chambers can be optimized separately to suit different enzyme requirements; highly efficient tandem catalysis: improving the efficiency of incompatible tandem reactions; and stable, ordered linkage: DNA phosphate groups form stable MOP bonds with the metal centers of MOFs, achieving ordered MOF linkage. Significantly improved performance: compared to disordered systems, the enzyme@core-satellite MOF system exhibits 1.5–2 times higher catalytic activity and 2–20 times higher thermal stability. Core advantages: spatially compartmentalized structure: supporting customized enzyme@core-satellite MOF design, enabling simultaneous incompatible tandem reactions within a single reactor; and ordered mass transfer regulation: promoting substrate / product transport and substrate recruitment, mitigating inhibition from high-concentration intermediate byproducts. This work provides a new paradigm for developing complex multi-enzyme catalytic compartment systems and has significant industrial application potential.

[0052] The innovations of this invention are as follows: 1) A gentle method is used to immobilize cascade enzymes in large and small metal-organic frameworks (MOFs), protecting the enzymes from external environmental influences. 2) A pioneering strategy utilizes DNA to link "core-satellite" MOFs. Stable MOP coordination bonds are formed between the DNA phosphate groups and the MOF metal center, allowing for the directional assembly of large-sized enzyme@MOF core units and small-sized enzyme@MOF satellite units, providing independent and tunable microenvironments for different enzymes. 3) This "core-satellite" structure enables substrate channeling, effectively mitigating the inhibitory effect of intermediate products (such as GlcA) on cascade enzymes (such as HRP), significantly improving catalytic efficiency. 4) The spatial separation characteristic of the "core-satellite" structure allows for the precise loading of incompatible cascade enzymes with mutual interference (such as active site competition and product cross-inhibition) into the core MOF and satellite MOF, respectively, maintaining independent functional conformations and microenvironments for both types of enzymes within the same system, achieving highly efficient synergistic catalysis. 5) Through spatial separation and MOF framework protection, the enzyme's thermal stability, solvent tolerance, and reusability are comprehensively improved, solving the problem of poor stability between free enzymes and traditional immobilization systems. 6) The assembly process is conducted under mild conditions (room temperature, acidic environment), and the DNA sequence allows for precise control of assembly efficiency and structure, facilitating large-scale production. System recovery can be achieved simply by centrifugation, simplifying the operation process and reducing industrial application costs. 7) This system is suitable for modular multi-enzyme configurations and can be flexibly adapted to various combinations of dual-enzyme and multi-enzyme systems, exhibiting excellent customizability and providing a universal platform for complex biocatalytic reactions. 8) This biomimetic, compartmentalized metabolic system, mimicking natural cells, achieves ordered spatial organization of enzymes through artificial design, providing a novel approach to constructing multi-compartment bioreactors and possessing significant theoretical and applied value.

[0053] The core-satellite system used in this invention can be applied to the immobilization of cascade enzymes. By utilizing an in-situ synthetic immobilization method, cascade enzymes are encapsulated in the "core" and "satellite" MOF chambers, respectively, constructing a spatially separated multi-enzyme catalytic system (Enzyme@core-satellite MOFs). Then, DNA is used to link the two types of MOFs, resulting in a large number of satellite MOFs uniformly attached to the surface of the core MOF, thus preparing a core-satellite enzyme-MOF composite catalytic material. This core-satellite enzyme-MOF composite catalytic material is then applied to the catalytic reactions of different cascade enzymes. The core-satellite enzyme-MOF composite catalytic material prepared by this invention can control the growth, particle size, and uniformity of satellite unit distribution on the core unit surface of both the core and satellite MOF units, thereby achieving the enrichment of satellite MOF units on the surface of the core MOF unit while ensuring enzyme activity. The composite material prepared using this invention is applicable to the catalytic systems of various biological enzymes, thereby improving system stability, reusability, operability, continuity, and ease of practical application.

[0054] The core-satellite metal-organic framework (MOF) bioenzyme microreactor composite material used in this invention is a hierarchically ordered composite material formed by self-assembly of a large-sized core MOF and a small-sized satellite MOF through MOP coordination bonds formed between the phosphate groups of DNA and the metal centers of the MOF. This is the first reported application of DNA MOP coordination to achieve hierarchical and ordered assembly of MOFs for multi-enzyme catalysis. Through DNA-guided directional assembly, the core MOF and satellite MOF are precisely connected to form a core-satellite enzyme-MOF system with independent microenvironments. The advantages of this system are: high cascade catalytic efficiency; significantly improved enzyme thermal stability, solvent tolerance, reusability, and storage stability; the porous structure of the core and satellite MOFs provides a large specific surface area, which is beneficial for substrate diffusion and catalytic reactions; compared with traditional single MOF immobilization systems, this system reduces enzyme interference through spatial separation, and the negative charge stabilization system provided by DNA reduces MOF disintegration, ensures enzyme structural stability, enables recycling, and thus reduces costs. The core-satellite system constructed by this invention can be applied to a variety of enzyme cascade reactions, improving the efficiency, stability and operability of complex catalytic processes.

[0055] This invention provides a DNA-linked core-satellite metal-organic framework (MOF) bioenzyme microreactor composite material. This composite material possesses the characteristics of being environmentally friendly and highly efficient in catalysis; it also inherits the advantages of enzyme-MOF materials, such as large specific surface area and good chemical stability. Compared to traditional MOF materials, the core-satellite MOF bioenzyme microreactor composite material constructed in this invention can precisely control the spatial arrangement of enzymes and adapt to the microenvironment, thereby achieving efficient incompatible cascade reactions while ensuring high enzyme activity. The system constructed using this invention can be applied to various complex enzyme catalytic systems, thereby improving the system's catalytic efficiency, stability, reusability, and ease of practical application.

[0056] In biological metabolic pathways, cascade enzyme synergistic catalysis is key to achieving efficient metabolism, but its large-scale application faces challenges such as the poor stability of natural enzymes and conflicting reaction conditions (e.g., pH / temperature). While metal-organic frameworks (MOFs) provide ideal carriers for multi-enzyme immobilization, they struggle to create microenvironmentally tunable, precisely ordered, and customizable multilayer enzyme reactors, limiting the efficient execution of complex cascade reactions. The "core-satellite" enzyme-MOF assembly in this study forms stable MOP bonds between the phosphate groups of DNA molecules and the metal centers of MOFs, directionally linking large-sized core MOFs and small-sized satellite MOFs into an ordered structure. This enables independent microenvironment customization, efficient tandem catalysis, and simultaneous execution of incompatible reactions, significantly improving catalytic activity and thermal stability. Furthermore, the ordered structure promotes mass transfer, avoiding byproduct inhibition caused by disorder in traditional MOF systems. In addition, optimization experiments, including customizing the enzyme immobilization location and DNA sequence, revealed a significant decrease in catalytic activity in non-optimized systems, primarily due to disordered spatial arrangement or microenvironmental mismatch, affecting multi-enzyme synergy and substrate transformation.

[0057] This invention connects two MOFs of different sizes via DNA and applies them to the catalysis of cascade enzymes. Because each cascade enzyme has different optimal conditions, it is often necessary to regulate the external environment (pH, temperature, etc.) in which the enzyme resides. Existing technologies use core-satellite MOF framework materials that can independently regulate and confine the microenvironment. The advantages of this invention are: firstly, it protects the enzyme from denaturation and inactivation; secondly, it allows for better catalytic activity of the cascade enzyme by regulating the enzyme's location in different compartments (due to differences in core / satellite or different MOF materials); thirdly, it effectively isolates the influence of byproducts; and fourthly, it provides a gentle assembly suitable for various cascade enzyme systems. This invention is the first to create a DNA-mediated core-satellite MOF framework and a microreactor composite material in which the enzyme is disassembled within the framework. Directed assembly and spatially separated enzyme loading are achieved through the MOP coordination bonds of DNA. It utilizes green and precise DNA assembly to replace stringent fixation conditions, and the core-satellite structure enables the compartmentalization of incompatible enzymes, overcoming the bottlenecks of enzyme inactivation, inter-enzyme interference, and untunable microenvironments in existing technologies.

[0058] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0059] 1. This invention innovatively utilizes the phosphate groups of DNA to form MOP coordination bonds with MOF metal nodes, constructing a "core-satellite" hierarchical structure, and for the first time achieving the directional assembly of large-size core MOFs and small-size satellite MOFs. This structure possesses high porosity, an independently controllable microenvironment, and an ordered spatial arrangement, providing independently adapted reaction sites for multiple enzymes, greatly preserving the enzyme's highly efficient catalytic activity, while overcoming the disordered spatial arrangement defects of traditional MOF systems. It significantly improves the enzyme's thermal stability, solvent tolerance, and storage stability, facilitates recovery and reuse, and reduces reaction costs.

[0060] 2. This invention uses in-situ synthesis to immobilize cascade enzymes in different MOFs, and uses DNA to precisely connect the core and satellite MOFs to form an ordered system. Compared with single MOF immobilized enzyme materials, this system can promote substrate channelization and delivery through spatial separation, reduce intermediate inhibition (such as the effect of GlcA on HRP), and simplify subsequent separation and recovery operations. The introduction of DNA increases the stability of the system, enabling long-term recycling and thus reducing costs.

[0061] 3. This core-satellite enzyme-MOF system can be applied to various multi-enzyme catalytic systems, such as the glucose oxidase & horseradish peroxidase (GOx & HRP) two-enzyme cascade reaction, the N-acetylglucosamine 1-kinase & uridine transferase (NahK & GlmU) glycosyl donor synthesis reaction, the Candida albicans lipase & cytochrome P450 enzyme (CRL & CYP450) hydroxy fatty acid synthesis reaction, and the β-galactosidase & glucose oxidase & horseradish peroxidase (β-Gal & GOx & HRP) three-enzyme cascade reaction. This demonstrates that the system possesses good biocompatibility, customizability, and operational continuity, and is compatible with enzyme cascades under different reaction conditions.

[0062] 4. The preparation process of this invention is mild (room temperature, acidic environment), and the DNA sequence and MOF material are widely available and cost-controllable. The constructed core-satellite enzyme-MOF system inherits the high efficiency of enzyme catalysis and the structural stability of MOF. It is environmentally friendly, easy to scale up for production, and can be widely used in industrial fields such as biosensing and biosynthesis. Attached Figure Description

[0063] Figure 1 A schematic diagram showing the relative catalytic activity of enzyme@MOF core units with different framework (ligand:metal) ratios;

[0064] Figure 2 A schematic diagram showing the relative catalytic activities of enzyme@MOF satellite units with different framework ratios (ICA:Ni);

[0065] Figure 3 A schematic diagram illustrating the relative catalytic activities of core-satellite enzyme@MOF composites assembled with different mass ratios (core:satellite);

[0066] Figure 4 A schematic diagram illustrating the relative activity of different acid solutions on the post-treatment core-satellite metal-organic framework bioenzyme microreactor composite material;

[0067] Figure 5 A schematic diagram illustrating the relative activity of post-treatment core-satellite metal-organic framework bioenzyme microreactor composite material with different pH acid solutions.

[0068] Figure 6 A schematic diagram illustrating the relative activity of different DNA types on the post-processing core-satellite metal-organic framework bioenzyme microreactor composite material;

[0069] Figure 7 A schematic diagram illustrating the relative activity of DNA of different lengths on the post-processing core-satellite metal-organic framework bioenzyme microreactor composite material;

[0070] Figure 8A schematic diagram illustrating the relative activity of different DNA concentrations on the post-processing core-satellite metal-organic framework bioenzyme microreactor composite material;

[0071] Figure 9 A schematic diagram showing the effect of different post-treatment times on the relative activity of the core-satellite metal-organic framework bioenzyme microreactor composite material;

[0072] Figure 10 A schematic diagram of temperature stability testing for GOx&HRP@core-satellite MOF;

[0073] Figure 11 A schematic diagram of the organic solvent resistance test for GOx&HRP@core-satellite MOF;

[0074] Figure 12 A schematic diagram of the stability test for recycling GOx&HRP@core-satellite MOF;

[0075] Figure 13 A schematic diagram of the storage utilization stability test for GOx&HRP@core-satellite MOF;

[0076] Figure 14 Schematic diagram of the catalytic activity of GOx & HRP@ different core-satellite MOF assemblies (Note: In GOx@ZIF-90+HRP@ZIF-90, the first ZIF-90 that appears is a large-sized core MOF, and the second ZIF-90 that appears is a small-sized satellite MOF. ZIF-8, ZIF-67 and Ni(ICA)2 are the same as ZIF-90).

[0077] Figure 15 The reaction equations are those involved in the experiment.

[0078] Figure 16 A schematic diagram of the relative activity of the GOx&HRP@core-satellite metal-organic framework bioenzyme microreactor composite material;

[0079] Figure 17 A schematic diagram of the relative activity of the NahK&GlmU@ core-satellite metal-organic framework bioenzyme microreactor composite material;

[0080] Figure 18 A schematic diagram of the relative activity of the CRL&CYP450@core-satellite metal-organic framework bioenzyme microreactor composite material;

[0081] Figure 19 A schematic diagram of the relative activity of the β-Gal&GOx&HRP@core-satellite metal-organic framework bioenzyme microreactor composite material;

[0082] Figure 20 HPLC chromatograms of different immobilization systems in the NahK&GlmU core-satellite metal-organic framework bioenzyme microreactor composite material during the synthesis of UDP-GlcNAc. Detailed Implementation

[0083] The present invention can be better understood from the following embodiments. Those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0084] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer. Specifically, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, (HOCH2)3CNH2), glucose (C6H2O)... 12 O6), CRL lipase from Candida albicans (purchased from Sigma Aldrich (Shanghai, China), catalog number 62301-5G-F); Glucose oxidase (GOx), 3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB) (purchased from Sangon Biotech Co., Ltd.); Horseradish peroxidase (HRP), β-galactosidase (β-Gal), adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate (UTP) (purchased from Shanghai Maclean Biotechnology Co., Ltd.); N-acetylaminohexose 1-kinase (NahK, ANC68241.1), uridine transferase (GlmU, ACO759) 77.1) Cytochrome P450 enzyme (CYP450, AB597883) was synthesized artificially or obtained commercially based on the sequence from NCBI; N,N-dimethylformamide (DMF), petroleum ether (PE), ethyl acetate (EA), methanol (MeOH), N-acetyl-D-glucosamine (GlcNAc), and uridine 5'-bisphosphate-N-acetylglucosamine sodium salt (UDP-GlcNAc) (purchased from Aladdin Reagent (Shanghai) Co., Ltd.); magnesium chloride (MgCl2) and α-lactose (purchased from Shanghai Husheng Chemical Co., Ltd.); Bradford protein assay kit for detecting 0.1–1.5 mg / mL protein (purchased from Beyotime Biotechnology Co., Ltd.); and the DNA sequence used in the experiment (synthesized by Sangon Biotech Co., Ltd.).

[0085] Example 1

[0086] Synthesis of core MOF units

[0087] A 1 mL, 1 mmol / L ligand solution and a 1 mL, 1 mmol / L metal salt solution were mixed thoroughly. The ligand solution was selected from one of 2-methylimidazole, 2-formaldehyde imidazole, 2-hydroxypyrimidine, and 2-hydroxy-5-fluoropyrimidine. The metal salt was selected from one of zinc nitrate, zinc acetate, and cobalt acetate. The reaction was carried out at 45 °C for 12 h with a magnetic stirrer at 300 rpm. After the reaction, a reaction solution containing the core MOF unit was obtained. After centrifugation and washing (8000 rpm, 6 min), the core MOF unit was obtained. The diameter of the core MOF unit was 3–20 μm.

[0088] Example 2

[0089] Synthesis of satellite MOF units

[0090] A 1 mL, 1 mmol / L ligand solution and a 1 mL, 1 mmol / L metal salt solution were mixed thoroughly. The ligand solution was selected from one of 2-methylimidazole, 2-formaldehyde imidazole, 2-hydroxypyrimidine, and 2-hydroxy-5-fluoropyrimidine. The metal salt was selected from one of zinc nitrate, zinc acetate, nickel nitrate, and cobalt acetate. The reaction was carried out at 45 °C for 1 h with a magnetic stirrer at 300 rpm. After the reaction, a reaction solution containing satellite MOF units was obtained. After centrifugation and washing (8000 rpm, 6 min), satellite MOF units with a diameter of 0.05–1 μm were obtained.

[0091] Example 3

[0092] Preparation method of enzyme@MOF core unit

[0093] A mixture of 1 mL of a 1 mmol / L ligand solution, 1 mL of a 1 mg / mL enzyme solution, and 1 mL of a 1 mmol / L metal salt solution was thoroughly mixed. The ligand solution was selected from one of 2-methylimidazole, 2-formaldehyde imidazole, 2-hydroxypyrimidine, and 2-hydroxy-5-fluoropyrimidine. The metal salt was selected from one of zinc nitrate, zinc acetate, and cobalt acetate. The enzyme solution was at least one of β-galactosidase (β-Gal), glucose oxidase (GOx), horseradish peroxidase (HRP), N-acetaminohexose 1-kinase (NahK), uridine transferase (GlmU), Candida albicans lipase (CRL), and cytochrome P450 enzyme (CYP450). The reaction was carried out at 45°C for 12 h using a magnetic stirrer at 300 rpm. After the reaction, a reaction solution containing enzyme@MOF core units was obtained. After centrifugation and washing (8000 rpm, 6 min), enzyme@MOF core unit composite materials with a diameter of 3~20 μm were obtained.

[0094] Following the above method, the synthesis conditions of MOF were optimized using ZIF-90 as a template (i.e., 2-methylimidazole was selected as the ligand, and zinc nitrate was selected as the metal salt). The enzyme concentration was kept constant, and the molar ratio of 2-formaldehyde imidazole to zinc nitrate was adjusted. The effects of the molar ratio of 2-formaldehyde imidazole to zinc nitrate being 1:4 to 8:1 were determined.

[0095] The system was optimized using lipase (CRL) from *Candida albicans* as the template enzyme. 10% (by mass) of the composite material was added to a suspension containing p-nitrobenzene palmitate (0.8 mL, 200 mmol / L) for esterification and hydrolysis of the lipase. The reaction was carried out in a metal bath at 30°C for 15 min at 700 rpm. After the reaction, the supernatant was collected by centrifugation (8000 rpm, 2 min), and the absorbance was measured at 406 nm using a microplate reader to calculate the relative activity. The relative activity of the optimal activity group within each group was set as 100%. The activity results are as follows: Figure 1 As shown, the final product CRL@MOF core unit exhibited optimal activity when the molar ratio of 2-formaldehyde imidazole to zinc nitrate was 6:1. Subsequent experiments were conducted using an enzyme@MOF core unit composite material prepared with a ligand-metal salt molar ratio of 6:1.

[0096] The definition of relative activity (%) is:

[0097]

[0098] Where A is the absorbance of the sample under optimized conditions, and B is the absorbance of the optimal reaction under the same group conditions.

[0099] Example 4

[0100] Preparation method of enzyme@MOF satellite unit

[0101] 1 mL of 1 mmol / L 2-formaldehyde imidazole solution, 1 mL of 1 mg / mL enzyme solution, and 1 mL of 1 mmol / L nickel nitrate hexahydrate solution were mixed thoroughly. The enzyme solutions were at least one of the following: β-galactosidase solution (β-Gal), glucose oxidase solution (GOx), horseradish peroxidase solution (HRP), N-acetaminohexose 1-kinase solution (NahK), uridine transferase solution (GlmU), Candida albicans lipase solution (CRL), and cytochrome P450 enzyme solution (P450). The mixture was stirred evenly on a magnetic stirrer at 45°C for 1 h at a stirring speed of 300 rpm. After the reaction, a reaction solution containing enzyme@MOF satellite units was obtained. After centrifugation and washing (8000 rpm, 6 min), enzyme@MOF satellite unit composite materials with a diameter of 0.05–1 μm were obtained.

[0102] Following the above method, the synthesis conditions of MOF were optimized using Ni(ICA)2 as a template (i.e., 2-formaldehyde imidazole was selected as the ligand, and nickel nitrate was selected as the metal salt). The enzyme concentration was kept constant, and the molar ratio of 2-formaldehyde imidazole to nickel nitrate was adjusted. The effects of the molar ratio of 2-formaldehyde imidazole to nickel nitrate being 1:4 to 8:1 were determined.

[0103] The system was optimized using lipase (CRL) from *Candida albicans* as the template enzyme. 10% (by mass) of the composite material was added to a suspension containing p-nitrobenzene palmitate (0.8 mL, 200 mmol / L) for esterification and hydrolysis of the lipase. The reaction was carried out in a metal bath at 30°C for 15 min at 700 rpm. After the reaction, the supernatant was collected by centrifugation (8000 rpm, 2 min), and the absorbance was measured at 406 nm using a microplate reader to calculate the relative activity. The relative activity of the optimal activity group within each group was set as 100%. The activity results are as follows: Figure 2 As shown, the final product CRL@MOF satellite unit exhibited optimal activity when the molar ratio of 2-formaldehyde imidazole to nickel nitrate was 4:1. Subsequent experiments were conducted using an enzyme@MOF satellite unit composite material prepared with a ligand-metal salt molar ratio of 4:1.

[0104] Example 5

[0105] The core of DNA connectivity: the construction of satellite-shaped metal-organic frameworks

[0106] 40 mg of the core MOF unit from Example 3 and 80 mg of the satellite MOF unit from Example 4 were placed in an acidic environment. Single-stranded DNA (pH 4, concentration 1 mol / L, volume 1 mL citric acid solution) was added, along with 300 μL of 10 mmol / L single-stranded DNA as a linker. The reaction was carried out at room temperature for 40 min. Through self-assembly, the satellite MOF unit was uniformly enriched on the surface of the core MOF unit, forming a reaction liquid containing a core-satellite metal-organic framework material. After washing three times with ultrapure water, the precipitate was dried to obtain the core-satellite metal-organic framework material.

[0107] Example 6

[0108] Construction of a DNA-Connecting Core-Satellite Metal-Organic Framework Bioenzyme Microreactor Composite Material

[0109] 40 mg of enzyme@MOF core unit from Example 3 (with a ligand-to-metal salt molar ratio of 6:1 during synthesis) and 40 mg of enzyme@MOF satellite unit from Example 4 (with a ligand-to-metal salt molar ratio of 4:1 during synthesis) were placed in an acidic environment (pH 4, concentration 1 mol / L, volume 1 mL citric acid solution). Single-stranded DNA (10 mmol / L, 300 μL of 12 adenine (12A) single-stranded DNA, reacted at room temperature for 40 min) was added. Through self-assembly, the enzyme@MOF satellite unit was uniformly enriched on the surface of the enzyme@MOF core unit, forming a core-satellite-shaped metal-organic framework bioenzyme microreactor composite material with a spatially separated enzyme system. After washing three times with ultrapure water, the precipitate was dried to obtain the core-satellite-shaped metal-organic framework bioenzyme microreactor composite material. Following the methods described in Examples 3 and 4 above, the enzyme@MOF core unit and enzyme@MOF satellite unit prepared from glucose oxidase (GOx) and horseradish peroxidase (HRP) respectively were used as templates to optimize the system. A composite material at 10% of the mass of the substrate glucose was added to a glucose solution for glucose detection. The glucose reaction solution was 0.4 mL, 10 mmol / L, the metal bath temperature was 45℃, the reaction time was 20 min, the rotation speed was 700 rpm, and the centrifugation speed was 8000 rpm for 6 min. The absorbance was measured at 652 nm using a microplate reader, and the relative activity was calculated. The relative activity of the group with the best activity was set as 100%. The control group without DNA post-treatment was prepared by mixing the enzyme@MOF core unit obtained in Example 3 with the enzyme@MOF satellite unit obtained in Example 4, replacing the DNA solution with ultrapure water, and washing three times with ultrapure water after the reaction to obtain a simple mixed system.

[0110] First, using ZIF-90 optimized in Example 3 as the core MOF unit and Ni(ICA)2 optimized in Example 4 as the satellite MOF unit, the addition amounts of the enzyme@MOF core unit and the enzyme@MOF satellite unit were optimized. Glucose oxidase (GOx) was encapsulated in the core MOF as the enzyme@MOF core unit, and horseradish peroxidase (HRP) was selected as the enzyme@MOF satellite unit. The mass ratio of the enzyme@MOF core unit and the enzyme@MOF satellite unit was adjusted, and the effects of mass ratios of 1:8 to 4:1 were measured. The activity results are as follows: Figure 3As shown, the synthesized core-satellite metal-organic framework (MOF) bioenzyme microreactor composite material exhibits optimal activity when the mass ratio of enzyme@MOF core units to enzyme@MOF satellite units is 1:2. Subsequent experiments were conducted using the core-satellite MOF bioenzyme microreactor composite material prepared with an enzyme@MOF core unit to enzyme@MOF satellite unit mass ratio of 1:2, i.e., 40 mg of enzyme@MOF core units and 80 mg of enzyme@MOF satellite units.

[0111] Next, using ZIF-90 optimized in Example 3 as the core MOF unit and Ni(ICA)2 optimized in Example 4 as the satellite MOF unit, the addition amount during connection was 40 mg for the core MOF unit and 80 mg for the satellite MOF unit. The following optimizations were performed:

[0112] (1) The post-processing acid environment of the system was optimized. Three solution systems were used: Tris-HCl, citric acid solution, and PBS solution, with a pH of 4, a concentration of 1 mol / L, and a volume of 1 mL. 10 mmol / L single-stranded DNA composed of 12 adenine atoms (12A) was used as the ligand. The post-processing (i.e., self-assembly) time at room temperature was 40 min. The activity results are as follows: Figure 4 The system exhibits optimal activity when the acidic environment is a citric acid solution.

[0113] (2) The pH (2-7) of the acidic environment was optimized in a 1 mol / L citric acid solution of 1 mL, using 300 μL of 10 mmol / L single-stranded DNA composed of 12 adenine atoms (12A) as the ligand; the post-treatment (i.e., self-assembly) time at room temperature was 40 min, and the activity results are as follows. Figure 5 The system exhibits optimal activity when the pH of the acidic environment is 4.

[0114] (3) Optimization of DNA and post-treatment time was achieved. The DNA consisted of single-stranded DNA composed of adenine, guanine, cytosine, and thymine; the DNA length was 8-24 deoxyribonucleotides; the DNA concentration was 0-20 mmol / L; and the self-assembly time at room temperature was 10-70 min. Under the optimal acidic post-treatment environment at room temperature, the type and length of DNA were optimized, and the results are as follows: Figure 6 , Figure 7 As shown, the system exhibited optimal activity when using 300 μL of single-stranded DNA consisting of 12 adenine atoms (12A) as the ligand. Figure 6As shown, the relative activity of the system is less than 50% without the addition of DNA, indicating that DNA plays an important ligation role. Under optimal post-treatment acidic conditions and with optimal DNA type and length, the DNA concentration and post-treatment time were further optimized, as shown below. Figure 8 and Figure 9 The results showed that the activity was optimal when the DNA concentration was 10 mmol / L, and the system exhibited the best activity when the post-treatment (i.e., self-assembly) time was 40 min.

[0115] Furthermore, by replacing different ligating agents, such as polyacrylic acid, sodium polyacrylate, and polyethylene glycol, it was found that the DNA ligation effect was the best and the activity was optimal.

[0116] This invention uses enzyme@MOF core units and enzyme@MOF satellite units prepared from glucose oxidase (GOx) and horseradish peroxidase (HRP) respectively as templates to optimize the system. The composite material, with a mass of 10% of the substrate glucose, is added to a glucose solution for glucose detection. The glucose reaction solution is 0.4 mL, 10 mmol / L, the metal bath temperature is 45℃, the reaction time is 20 min, the rotation speed is 700 rpm, the centrifugation speed is 8000 rpm, and the time is 6 min. The absorbance is measured at 652 nm using an ELISA reader, and the relative activity is calculated. The relative activity of the best activity group in the same group is set as 100%.

[0117] Based on the above conclusions, the optimal preparation conditions are: take 40 mg of core MOF units and 80 mg of satellite MOF units, use 300 μL of 10 mmol / L single-stranded DNA composed of 12 adenine (12A) strands as a ligand, and treat with citric acid solution at pH 4 at room temperature for 40 min (i.e., self-assembly). The core-satellite metal-organic framework bioenzyme microreactor composite material prepared under these conditions is the optimized material.

[0118] All other materials were synthesized under the above optimal conditions: N-acetylglucosamine 1-kinase & uridine transferase-core-satellite metal-organic framework composite material (NahK&GlmU@core-satellite MOF), Candida albicans lipase & cytochrome P450 enzyme-core-satellite metal-organic framework composite material (CRL&CYP450@core-satellite MOF), and β-galactosidase & glucose oxidase & horseradish peroxidase-core-satellite metal-organic framework composite material (β-Gal&GOx&HRP@core-satellite MOF).

[0119] Example 7

[0120] Temperature stability test of DNA-directed assembly core-satellite enzyme-metal-organic framework system

[0121] A DNA-directed assembly core-satellite enzyme-metal-organic framework system was prepared according to the optimal method in Example 6. Enzyme@MOF core units and enzyme@MOF satellite units prepared with glucose oxidase (GOx) and horseradish peroxidase (HRP), respectively, were used as templates for temperature stability testing. The composite material, at 10% of the mass of the substrate glucose, was added to a glucose solution for glucose detection. The metal bath temperature was 45°C, the reaction time was 20 min, the rotation speed was 700 rpm, and the centrifugation speed was 8000 rpm for 6 min. The supernatant was collected, and the absorbance was measured at 652 nm using a microplate reader to calculate its relative activity. The relative activity of the optimal activity group within the same group was set as 100%. The stability of the glucose oxidase & horseradish peroxidase@core-satellite metal-organic framework composite material (GOx & HRP@core-satellite MOF) was determined at different temperatures (30°C, 40°C, 50°C, 60°C, 70°C). The temperature stability results are as follows: Figure 10 As shown, compared to the simple mixing system (without adding DNA, large and small MOFs are directly mixed according to the method in Example 6, and all other steps are the same), GOx&HRP@core-satellite MOFs maintain good stability in the range of 30℃ to 70℃, and exhibit the best activity at 40℃, which is consistent with the optimal activity temperature of the mixed system.

[0122] Example 8

[0123] Organic solvent stability test of a DNA-directed assembly core-satellite enzyme-metal-organic framework system

[0124] A DNA-directed assembly core-satellite enzyme-metal-organic framework system was prepared according to the optimal method in Example 6. Temperature stability was tested using enzyme@MOF core units and enzyme@MOF satellite units prepared from glucose oxidase (GOx) and horseradish peroxidase (HRP), respectively, as templates. A 10% (by mass) composite material of the substrate glucose was added to a glucose solution for glucose detection. The metal bath temperature was 45°C, the reaction time was 20 min, the rotation speed was 700 rpm, and the centrifugation speed was 8000 rpm for 6 min. The supernatant was collected, and the absorbance was measured at 652 nm using a microplate reader to calculate its relative activity. The relative activity of the optimal activity group within the same group was set as 100%. The stability of the glucose oxidase & horseradish peroxidase@core-satellite metal-organic framework composite material (GOx & HRP@core-satellite MOF) in different organic solvents (DMF, PE, EA, MeOH, 40°C) was determined. The composite material was incubated in different organic solvents for 20 min, washed three times with water, and then its activity was tested according to the above method. Organic solvent stability results are as follows Figure 11 As shown, compared to a simple mixing system (where DNA is not added, large and small MOFs are directly mixed according to the method in Example 6, and all other steps are the same), GOx&HRP@core-satellite MOFs maintain good stability in DMF, PE, EA, and MeOH.

[0125] Example 9

[0126] Recycling stability test of a DNA-directed assembly core-satellite enzyme-metal-organic framework system

[0127] A core-satellite enzyme-metal-organic framework system assembled from DNA was prepared according to the optimal method described in Example 6. The enzyme@MOF core unit and enzyme@MOF satellite unit, prepared using glucose oxidase (GOx) and horseradish peroxidase (HRP) respectively, were used as templates for temperature stability testing. A glucose detection reaction was performed by adding 10% (by weight of glucose) of the composite material to a glucose solution. The metal bath temperature was 45°C, the reaction time was 20 min, the rotation speed was 700 rpm, and the centrifugation speed was 8000 rpm for 6 min. The supernatant was collected, and the absorbance was measured at 652 nm using a microplate reader to calculate its relative activity. The relative activity of the optimal activity group within the same group was set as 100%. The recycling stability of the glucose oxidase & horseradish peroxidase@core-satellite metal-organic framework composite (GOx & HRP@core-satellite MOF) was determined separately. At pH 7 and 40℃, the GOx & HRP@core-satellite MOF composite was removed from the reaction system by centrifugation, washed three times with water, and then added to a fresh reaction solution for the next round of recycling stability testing. The recycling stability is as follows: Figure 12 As shown, GOx&HRP@core-satellite MOF retains more than 60% relative activity after being recycled 7 times.

[0128] Example 10

[0129] Storage stability test of a DNA-directed assembly core-satellite enzyme-metal-organic framework system

[0130] A DNA-directed assembly core-satellite enzyme-metal-organic framework system was prepared according to the optimal method in Example 6. Enzyme@MOF core units and enzyme@MOF satellite units prepared with glucose oxidase (GOx) and horseradish peroxidase (HRP), respectively, were used as templates for temperature stability testing. The composite material, at 10% of the mass of the substrate glucose, was added to a glucose solution for glucose detection. The metal bath temperature was 45°C, the reaction time was 20 min, the rotation speed was 700 rpm, and the centrifugation speed was 8000 rpm for 6 min. The supernatant was collected, and the absorbance was measured at 652 nm using a microplate reader to calculate its relative activity. The relative activity of the best activity group within the same group was set as 100%. The stability of the glucose oxidase & horseradish peroxidase@core-satellite metal-organic framework composite material (GOx & HRP@core-satellite MOF) after storage (0 days, 3 days, 6 days, 9 days, 12 days, 15 days, 18 days, 21 days) was measured. Storage stability is as follows: Figure 13 As shown, GOx&HRP@core-satellite MOFs still exhibit high activity after 21 days of storage.

[0131] Example 11

[0132] Practical Applications of Glucose Oxidase & Horseradish Peroxidase-Core-Satellite Metal-Organic Framework Composites (GOx & HRP@Core-Satellite MOF)

[0133] Glucose oxidase & horseradish peroxidase@core-satellite metal-organic framework composite material (GOx & HRP-core-satellite metal-organic framework bioenzyme microreactor composite material) was prepared according to the optimal method in Example 6. Representative ZIF-90, ZIF-8 (i.e., ligand selected as 2-methylimidazole, metal salt selected from zinc acetate), and ZIF-67 (i.e., ligand selected as 2-methylimidazole, metal salt selected from cobalt acetate) were selected as core MOF units, and ZIF-90, ZIF-8, ZIF-67, and Ni(ICA)2 were selected as satellite MOF units. The positions of the model enzyme encapsulated in the core unit and satellite unit were interchanged. A GOx&HRP-core-satellite metal-organic framework bioenzyme microreactor composite material (10% by weight of the substrate (glucose)) was added to the glucose reaction solution for glucose (0.4 mL, 10 mmol / L) detection. The reaction time was 20 min, the rotation speed was 700 rpm, and the centrifugation speed was 8000 rpm for 6 min. The absorbance was measured at 652 nm using a microplate reader, and the relative activity was calculated. The relative activity of the best-performing group within the same group was set as 100%. The activity graph is shown below. Figure 14 As shown, it can be seen that encapsulating the first step of the cascade enzyme reaction in the core MOF unit can increase the activity by about 2 times. That is, it is best to encapsulate glucose oxidase in the core MOF. Furthermore, ZIF-90 was selected as the core MOF unit and Ni(ICA)2 as the satellite MOF unit, resulting in the best overall activity. The following examples were all synthesized under optimal conditions.

[0134] The relative activity was calculated with equal amounts of the simple mixed group (large and small MOFs directly mixed without DNA, as described in Example 6) as 100%. The specific reaction process is as follows: Figure 15 As shown in Figure a. The reaction results are as follows. Figure 16 As shown, GOx&HRP@core-satellite MOF exhibits 178% higher activity compared to the simple mixture. This further demonstrates that the composite material prepared in this invention not only provides protection for the enzyme but also effectively enhances its enzyme activity.

[0135] Example 12

[0136] Practical Applications of N-acetaminohexose 1-position kinase & uridine transferase-core-satellite metal-organic framework composites (NahK & GlmU@core-satellite MOF)

[0137] The NahK & GlmU core-satellite metal-organic framework (MOF) bioenzyme microreactor composite material was prepared according to the optimal method described in Example 11. Using enzyme@MOF core units and enzyme@MOF satellite units prepared from N-acetylglucosamine 1-kinase NahK and uridine transferase GlmU, respectively, as templates, 10% (by mass) of the NahK & GlmU core-satellite MOF bioenzyme microreactor composite material was added to a 100 mmol / L Tris-HCl buffer solution (pH 7.5) containing ATP (10 mmol / L), UTP (10 mmol / L), monosaccharide substrate GlcNAc (10 mmol / L), and MgCl2 (2 mmol / L). The reaction was carried out at 37°C for 24 h. The glycosyl donor (uridine diphosphate-N-acetylglucosamine) was detected by high-performance liquid chromatography (HPLC) and mass spectrometry (MS / MS). HPLC conditions: Inertsil ODS-3 column (150 mm × 4.6 mm, 5 μm). Mobile phase A was a phosphate buffer solution consisting of 18 mmol / L sodium dihydrogen phosphate and disodium hydrogen phosphate (pH adjusted to 6.8 with phosphate), and mobile phase B was pure methanol. The sample pan temperature and chromatographic temperature were both 25°C. The detection wavelength was 272 nm, and the injection volume was 20 μL. Mass spectrometry detection of glyconucleotides was performed in negative ion mode. The HPLC results are shown below. Figure 20 As shown, it can be seen that there is a difference in activity between placing the first-step enzyme NahK in the core MOF and the second-step enzyme GlmU in the satellite MOF, and placing the first-step enzyme NahK in the satellite MOF and the second-step enzyme GlmU in the core MOF. Placing the first-step enzyme NahK in the core MOF and the second-step enzyme GlmU in the satellite MOF increases the catalytic activity by 20 times, proving that the system can be customized according to different enzymes. The specific reaction process is as follows... Figure 15 As shown in b. The reaction results are as follows. Figure 17 As shown, NahK&GlmU@core-satellite MOFs exhibit 155% higher activity than the simple mixture group (large and small MOFs directly mixed without DNA, as described in Example 6). This further demonstrates that the composite material prepared in this invention not only provides protection for the enzyme but also effectively enhances its enzyme activity.

[0138] The 1H NMR spectra of the glyconucleotide UDP-GlcNAc synthesized from N-acetaminohexose 1-kinase and uridyltransferase in Example 12 are as follows: 1H NMR (400 MHz, D2O) δ 7.97 (d, J = 7.9 Hz, 1H), 5.97 (dd, J = 8.1, 6.4 Hz, 2H), 5.53 (dd, J = 7.1, 3.2 Hz, 1H), 4.35 - 4.36 (m, 2H), 4.32 - 4.42 (m, 1H), 4.25 (ddd, J = 11.5, 4.4, 2.3 Hz, 1H), 4.20 (ddd, J = 11.5, 5.4, 3.2 Hz, 1H). Hz, 1H), 4.01 (dt, J=10.3, 3.0Hz, 1H), 3.95 (ddd, J=10.7, 4.4, 2.3 Hz, 1H), 3.93 - 3.96 (m, 1H), 3.84 - 3.80 (m, 2H), 3.57 (dd, J=10.0, 9.3 Hz, 1H), 2.09 (s, 3H).

[0139] Example 13

[0140] Practical Applications of Lipase & Cytochrome P450 Enzyme@Core-Satellite Metal-Organic Framework Composites (CRL & CYP450@Core-Satellite MOF) from Candida albicans

[0141] The core-satellite metal-organic framework (MOF) CRL & P450 bioenzyme microreactor composite material was prepared according to the optimal method in Example 11. Enzyme@MOF core units and enzyme@MOF satellite units prepared from Candida albicans lipase (CRL) and cytochrome P450 enzyme (CYP450), respectively, were used as templates. 10% (by weight) of the core-satellite MOF CRL & CYP450 bioenzyme microreactor composite material (p-nitrophenyl laurate) was added to 1.5 mL of a 2 mmol / L p-nitrophenyl laurate reaction solution for hydrolysis and hydroxylation. The H₂O₂ concentration was 30 mmol / L, the metal bath temperature was 25 °C, the reaction time was 36 h, and the rotation speed was 700 rpm. After the reaction, the mixture was extracted with ethyl acetate under acidic conditions and centrifuged at 8000 rpm for 6 min. The supernatant was collected and analyzed by gas chromatography-mass spectrometry (GC-MS) for the detection of p-hydroxylauric acid. Gas chromatography-mass spectrometry (GC-MS) conditions: initial temperature 100℃, held for 0.5 min, heating rate 10℃ / min, to 300℃. Helium gas flowed through the column at 0.7 mL / min. Injection temperature: 250℃. Split ratio: 99:1. Ionization method: electron impact ionization (70 eV). Mass scan range: m / z 33–400. The specific reaction process is as follows... Figure 15 As shown in c. The first-step enzyme CRL of the cascade is placed in the core MOF, and the second-step enzyme CYP450 is placed in the satellite MOF. The reaction result is as follows. Figure 18 As shown, CRL&P450@core-satellite MOF exhibited 134% more activity than the simple mixture group (large and small MOFs directly mixed without DNA, as described in Example 6). This further demonstrates that the composite material prepared in this invention not only provides protection for the enzyme but also effectively enhances its enzyme activity.

[0142] Example 14

[0143] Practical Applications of β-Galactosidase, Glucose Oxidase, Horseradish Peroxidase-Core-Satellite Metal-Organic Framework Composites (β-Gal, GOx, HRP @ Core-Satellite MOF)

[0144] The β-Gal&GOx&HRP core-satellite metal-organic framework (MOF) bioenzyme microreactor composite material was prepared according to the optimal method in Example 11. Using enzyme@MOF core units and enzyme@MOF satellite units prepared from glucose oxidase (GOx), β-galactosidase (β-Gal), and horseradish peroxidase (HRP) as templates, 10% (by mass) of the β-Gal&GOx&HRP core-satellite MOF bioenzyme microreactor composite material of the substrate (lactose) was added to a lactose reaction solution for lactose (0.4 mL, 10 mmol / L) detection. The reaction time was 20 min, the rotation speed was 700 rpm, and the centrifugation speed was 8000 rpm for 6 min. The absorbance was measured at 652 nm using a microplate reader, and the relative activity was calculated. The relative activity was calculated with a disordered system set at 100%. The specific reaction process is as follows... Figure 15 As shown in d. Encapsulating the second step of the cascade enzyme reaction (i.e., GOx) in the core MOF unit, and the first and third steps (i.e., β-Gal and HRP) in the satellite MOF unit, facilitates substrate diffusion. The reaction results are shown in... Figure 19 As shown, the β-Gal&GOx&HRP@core-satellite MOF encapsulated using the optimal method exhibited 162% more activity than the simple mixture group (large and small MOFs directly mixed without DNA, as described in Example 6). This further demonstrates that the composite material prepared in this invention not only provides protection for the enzyme but also effectively enhances its enzyme activity.

Claims

1. A core-satellite metal-organic framework connected by DNA, characterized in that, The core-satellite metal organic framework comprises: large-size core MOF units, small-size satellite MOF units, bridging DNA, which forms M-O-P coordination bonds with metal centers on the MOF through phosphate groups of the DNA, and which directionally connects the core MOF and the satellite MOF to form a core-satellite metal organic framework material.

2. The DNA-linked core-satellite metal-organic framework of claim 1, wherein, The core MOF unit has a diameter of 3-20 μm, and the satellite MOF unit has a diameter of 0.05-1 μm; the assembly formed by the core MOF unit and the satellite MOF unit connected by the DNA has a core-satellite structure, and a single core MOF unit is connected to multiple satellite MOF units; the bridging DNA has a length of 8-24 deoxyribonucleotides and is single-stranded DNA composed of any one or more of adenine, guanine, cytosine and thymine.

3. The DNA-linked core-satellite metal-organic framework of claim 1, wherein, The core MOF unit preferably uses one of zeolite imidazole framework materials and zeolite pyrimidine framework materials as a carrier material, and the satellite MOF unit uses a metal organic framework material with a diameter smaller than that of the core MOF unit as a carrier material.

4. A DNA-linked core-satellite metal-organic framework bioenzyme microreactor composite material, characterized in that, The composite material uses the core-satellite metal organic framework of claim 1 as a carrier, encapsulates different enzymes in the core MOF unit and the satellite MOF unit respectively to form a core-satellite metal organic framework biological enzyme microreactor composite material.

5. The DNA-linked core-satellite MOF bioenzyme microreactor composite material of claim 4, wherein, The enzymes include at least two of β-galactosidase (β-Gal), glucose oxidase (GOx), horseradish peroxidase (HRP), N-acetylhexosamine 1-kinase (NahK), uridine transferase (GlmU), Candida rugosa lipase (CRL) and cytochrome P450 enzyme (CYP450).

6. A method of preparing the DNA-linked core-satellite MOF bioenzyme microreactor composite material of claim 4, wherein, The method comprises the following steps: (1) synthesis of enzyme@MOF core unit: mixing and stirring a ligand solution, an enzyme solution and a metal ion solution to obtain a reaction solution of enzyme@MOF composite catalytic material, and centrifuging to obtain enzyme@MOF core unit composite material; (2) synthesis of enzyme@MOF satellite unit: mixing and stirring a ligand solution, an enzyme solution and a metal ion solution to obtain a reaction solution of enzyme-metal organic framework composite catalytic material, and centrifuging to obtain enzyme@MOF satellite unit composite material; (3) DNA directional assembly: placing the core unit and the satellite unit in an acidic environment and adding bridging DNA to obtain a core-satellite metal organic framework biological enzyme microreactor composite material through self-assembly.

7. The production method according to claim 6, characterized by, In step (1), the ligand is one of 2-methylimidazole, 2-formaldehyde imidazole, 2-hydroxypyrimidine and 2-hydroxy-5-fluoropyrimidine, the salt of the metal ion is one of zinc nitrate, zinc acetate and cobalt acetate, the molar ratio of the ligand to the metal ion is 1:4-8:1, the enzyme content in the ligand solution, the enzyme solution and the metal ion solution system is 1-4 mg / mL, the reaction temperature is 40-50℃, the stirring speed is 200-400 rpm, and the reaction time is 10-15 h.

8. The preparation method according to claim 6, characterized in that, The ligand in step (2) is one of 2-methylimidazole, 2-formaldehyde imidazole, 2-hydroxy pyrimidine and 2-hydroxy-5-fluoropyrimidine, the salt of metal ion is one of zinc nitrate, zinc acetate, nickel nitrate and cobalt acetate, the molar ratio of the ligand to the metal ion is 1:4-8:1, the enzyme content in the ligand solution, enzyme solution, metal ion solution system is 1-4 mg / mL, the reaction temperature is 40-50°C, the stirring is 200-400 rpm, and the reaction time is 0.5-2 h.

9. The preparation method according to claim 6, characterized in that, In step (3), the acidic environment is a Tris-HCl solution, a citric acid solution or a PBS solution with a concentration of 0.2-2 mol / L and a pH of 4-6; the enzyme@MOF core unit composite material is 30-50 mg; the enzyme@MOF satellite unit is 60-100 mg; the DNA concentration is 10-15 mmol / L, and the volume is 200-500 μL; the self-assembly time is 10-70 min, and the temperature is room temperature.

10. Application of the DNA-linked core-satellite metal organic framework biological enzyme micro-reactor composite material in different multi-enzyme cascade catalytic systems.

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