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CN-121975790-A - Heterogeneous core-shell double-enzyme cascade reactor and preparation method and application thereof

CN121975790ACN 121975790 ACN121975790 ACN 121975790ACN-121975790-A

Abstract

The invention provides a heterogeneous core-shell double-enzyme cascade reactor and a preparation method and application thereof, belonging to the technical field of sensing or photocatalysis. The preparation method of the heterogeneous core-shell double-enzyme cascade reactor comprises the steps of mixing and incubating a first enzyme and a carboxylated metal-organic framework in a room temperature water phase, washing to obtain a metal-organic framework heterogeneous seed crystal loaded with the first enzyme, dispersing the heterogeneous seed crystal in an aqueous solution of an amine monomer for preassembling, adding an aldehyde monomer, a second enzyme and an ionic liquid catalyst into the obtained system, reacting at room temperature, separating and washing solids after the reaction is finished, and obtaining the heterogeneous core-shell double-enzyme cascade reactor. The invention aims to realize high activity retention of enzyme in a heterogenous framework through a mild and biocompatible interface guiding assembly strategy, solve the problems of mass transfer regulation and structural stability of a single porous material, and remarkably improve cascade reaction efficiency and operation stability.

Inventors

  • SHI JIE
  • ZHAO BAOZHU
  • CHENG ZHIFEI
  • XU BAOCAI

Assignees

  • 合肥工业大学

Dates

Publication Date
20260505
Application Date
20260211

Claims (10)

  1. 1. The preparation method of the heterogeneous core-shell double-enzyme cascade reactor is characterized by comprising the following steps of: (1) Mixing and incubating the first enzyme and the carboxylated metal-organic framework in a room temperature water phase, and washing the obtained solid to obtain a heterogeneous seed crystal of the metal-organic framework loaded with the first enzyme; (2) Dispersing the heterogeneous seed crystal obtained in the step (1) in an aqueous solution of an amine monomer, and pre-assembling; (3) Adding an aldehyde monomer, a second enzyme and an ionic liquid catalyst into the system obtained in the step (2), reacting at room temperature, separating and washing solids after the reaction is finished, and obtaining the heterogeneous core-shell double-enzyme cascade reactor.
  2. 2. The method for preparing a heterogeneous core-shell double enzyme cascade reactor according to claim 1, wherein in the step (1), the carboxylated metal organic framework is (COOH) 2 -UIO-66, and the first enzyme is glucose oxidase.
  3. 3. The method for preparing a heterogeneous core-shell double enzyme cascade reactor according to claim 1, wherein in the step (1), the incubation time is 1 hour.
  4. 4. The method for preparing a heterogeneous core-shell double enzyme cascade reactor according to claim 1, wherein in the step (2), the amine monomer is p-phenylenediamine, and the dosage ratio of the heterogeneous seed crystal to the amine monomer is 4 mg/6.4 mg.
  5. 5. The method for preparing a heterogeneous core-shell double enzyme cascade reactor according to claim 1, wherein in the step (3), the aldehyde monomer is 1,3, 5-trimethylbenzene, the ionic liquid catalyst is [ HOOCMMIM ] [ Cl ], and the second enzyme is horseradish peroxidase.
  6. 6. The method for preparing a heterogeneous core-shell double enzyme cascade reactor according to claim 1, wherein the dosage ratio of the heterogeneous seed crystal to the aldehyde monomer, the second enzyme and the ionic liquid catalyst is 4 mg/6.4 mg/2 mg/10 μl.
  7. 7. The method for preparing a heterogeneous core-shell double enzyme cascade reactor according to claim 1, wherein in the step (3), the reaction time is 10 minutes.
  8. 8. A heterogeneous core-shell double-enzyme cascade reactor, characterized in that the reactor is prepared by the preparation method according to any one of claims 1-7, and the reactor is a microsphere with a core-shell structure, comprising: an inner catalytic core composed of a carboxylated metal organic framework and a first enzyme; And an outer catalytic shell composed of a covalent organic framework and a second enzyme and coating the inner catalytic core.
  9. 9. The heterogeneous core-shell dual enzyme cascade reactor of claim 8, wherein the heterogeneous core-shell dual enzyme cascade reactor has an average particle size of 410nm.
  10. 10. Use of the heterogeneous core-shell dual enzyme cascade reactor of claim 8 or 9 for the preparation of a glucose biosensor.

Description

Heterogeneous core-shell double-enzyme cascade reactor and preparation method and application thereof Technical Field The invention belongs to the technical field of sensing or photocatalysis, and particularly relates to a heterogeneous core-shell double-enzyme cascade reactor, and a preparation method and application thereof. Background With the accelerated transformation of manufacturing industry to green, efficient and sustainable modes, enzymes as the core of biological manufacturing have shown significant potential in the fields of biological medicine, environmental remediation, food processing and the like. The enzyme immobilization technology is proved to be an effective strategy for improving enzyme stability and reusability, and lays a foundation for designing a multi-enzyme cascade system. The system can avoid intermediate product separation and inhibit side reaction by integrating continuous catalysis steps, thereby greatly improving the overall catalysis efficiency, however, the key point for realizing the efficient collaborative catalysis is that the carrier material needs to have accurate space organization capacity and efficient mass transfer regulation performance. Porous organic framework materials, particularly Metal Organic Frameworks (MOFs) and Covalent Organic Frameworks (COFs), have become important carriers for immobilization of mono-or multi-enzymes by virtue of their high designability of pore size, structure and surface properties. In particular MOFs, which can provide adjustable positioning environment, metal-enzyme synergy and mild embedding conditions to realize high loading and synergistic catalysis of enzymes, the inherent microporous structure of the traditional MOFs severely limits the mass transfer efficiency of biomacromolecules, and meanwhile, the hydrophobic surface, active ligand and water stability of partial MOFs (especially ZIF series) are insufficient, so that conformational inactivation of enzymes is extremely easy to induce. Although the existing strategies such as construction of hierarchical pore structures or post-synthesis modification can improve diffusion efficiency to a certain extent, the existing strategies are still subject to complicated preparation and insufficient universality, and lack of an integrated scheme capable of synergistically overcoming the contradiction between mass transfer limitation and stability. On the other hand, COFs become an ideal platform for in-situ enzyme encapsulation by virtue of strong covalent bond connection and excellent structural stability, however, traditional imino COFs are mostly synthesized by solvent heat, and the related organic solvents and harsh conditions such as strong acid are extremely easy to cause enzyme inactivation, and deviate from the mild environment required by biological macromolecules. Although green synthesis methods such as a step strategy of 'pre-protection-post crystallization' or room temperature mechanical grinding are presented in recent years, the above strategies are still limited by chemical incompatibility of synthesis media, mechanical force induced conformational damage and insufficient preparation controllability, and severely restrict the realization of high-performance enzyme encapsulation. Inspired by the realization of multienzyme cooperation in nature through organelle space separation, the construction of an artificial multienzyme cascade micro-compartment is a feasible method for protecting enzymes from environmental influences and keeping good functions and efficient mass transfer of the enzymes. In order to overcome the bottleneck of a single traditional porous material, the heterogeneous multi-layer structure (HMA) can have the advantages of all components by strategically integrating different types of porous materials, and provides a promising solution idea. Although HMA structures have demonstrated excellent performance in the sensing or photocatalytic fields, no report has been made to construct artificial multi-enzyme cascade micro-compartments based thereon. This is mainly due to the harsh synthesis conditions typically required for heterogeneous multi-layer frameworks, the susceptibility to enzyme inactivation, and the extremely difficult realization of precise spatial localization of multiple enzymes in complex structures. Therefore, the prior art still faces a core challenge in the aspects of heterostructure integration and enzyme space adaptation, and development of a mild and biocompatible interface-oriented assembly strategy is needed to cooperatively solve the problems of synthesis incompatibility and space adaptation, so as to realize controllable preparation of a bionic multi-enzyme cascade micro-compartment with high bioactivity retention, excellent structural stability and a hierarchical mass transfer channel. Despite the advances made in enzyme immobilization and cascade reactor construction in the prior art, there are a number of challenges that are diffic