CN-115536751-B - Method for guiding efficient and universal enzyme coating through seed crystal and application thereof

Abstract

The invention belongs to the technical field of metal-organic framework (MOF) immobilized enzymes, and particularly relates to a method for efficiently and universally coating enzymes through seed crystal guiding and application thereof. The invention discloses a method for synthesizing an enzyme@MOF compound efficiently and universally through seed crystal guiding, which is based on the characteristic of MOF crystal growth, creatively provides a seed crystal guiding synthesis method, wherein the addition of seed crystals can greatly promote the synthesis efficiency of the enzyme@MOF compound, greatly increase the encapsulation efficiency of the enzyme, and meanwhile, the seed crystal guiding synthesis method has no requirement on the chemical properties of the enzyme/protein, does not need destructive modification of the enzyme/protein to achieve the effect of improving the encapsulation efficiency, has good universality, solves the problems of low universality and low efficiency in the enzyme@MOF compound synthesis method at the present stage, and has promising application prospects in the fields of biocatalysis, biosensing, biological medicine and the like.

Inventors

• YANG TAO
• HUANG ZHIYANG
• ZENG HUI

Assignees

• 中山大学

Dates

Publication Date

20260508

Application Date

20220802

Claims (2)

1. 1. A method for guiding a high-efficiency universal synthetase@MOF compound through seed crystals is characterized in that in the process of encapsulating the enzyme by using the MOF, a slow nucleation stage is skipped by adding seed crystals corresponding to the MOF so as to achieve the purpose of rapidly synthesizing the synthetase@MOF compound; the method for directing the efficient and universal synthetase@MOF complex through seed crystals is as follows: S1, stirring 2-methylimidazole solution and zinc acetate solution at room temperature for reaction to obtain seed crystals, wherein the stirring reaction time is 12-36h, the concentration of the 2-methylimidazole solution is 2000-3000 mM, the concentration of the zinc acetate solution is 30-50 mM, and the volume ratio of the 2-methylimidazole solution to the zinc acetate solution is 1:1; S2, mixing a seed crystal solution and an enzyme solution, adding a 2-methylimidazole solution and a zinc acetate solution, and standing at room temperature to obtain an enzyme@MOF compound, wherein the concentration of the seed crystal solution is 4-6 mg/mL, the enzyme comprises cytochrome C, hemoglobin and myoglobin, the MOF is a ZIF-8 metal organic framework, the standing reaction time is 3-5h, the concentration of the enzyme solution is 0.5-2 mg/mL, the concentration of the 2-methylimidazole solution is 1200-1600 mM, the concentration of the zinc acetate solution is 90-110 mM, and the volume ratio of the seed crystal solution, the enzyme solution, the 2-methylimidazole solution and the zinc acetate solution is 1:1:2:2.
2. 2. Use of the method of claim 1 for the synthesis of MOF immobilized enzymes.

Description

Method for guiding efficient and universal enzyme coating through seed crystal and application thereof Technical Field The invention belongs to the technical field of metal-organic framework (MOF) immobilized enzymes, and particularly relates to a method for efficiently and universally coating enzymes through seed crystal guiding and application thereof. Background The enzyme is a natural catalyst with high selectivity and high activity, has the potential of improving catalytic efficiency, selectivity and environmental sustainability, and is widely applied to the fields of industrial catalysis, chemical synthesis, biotechnology and the like. However, the stability of enzymes after leaving the cell is poor, and is susceptible to inactivation by pH, temperature, most organic solvents and inhibitors, thereby greatly affecting the extracellular use of enzymes. Thus, increasing the stability of enzymes is a critical issue that is currently in need of resolution. In this regard, researchers have proposed strategies for immobilizing enzymes using metal-organic frameworks (MOFs) that take advantage of the variety diversity, high porosity and specific surface area, good stability, easily-adjustable pore size, and mild synthetic conditions of metal-organic frameworks to provide effective protection for enzymes. MOF immobilized enzymes can be carried out by surface biological binding, permeation, encapsulation, and the like. Wherein, surface biological binding and permeation belongs to a strategy of synthesizing MOF and then immobilizing enzyme, and encapsulation belongs to a strategy of synthesizing MOF and immobilizing enzyme simultaneously. Studies have shown that the direct encapsulation of enzymes into MOFs provides excellent protection and is therefore an important area of current research. The MOF enzyme encapsulating method adopts a one-pot synthesis method as a main research method, wherein the solution phase synthesis method mainly comprises a coprecipitation method and a biomimetic mineralization method. The coprecipitation method relies on spontaneous nucleation of MOFs, which encapsulates enzymes precipitated onto the surface of MOFs during nucleation and growth of MOFs, but is limited by the slowness of the spontaneous nucleation process, which is time consuming and inefficient. In the biomimetic mineralization method, the enzyme participates in the nucleation process, so that the synthesis efficiency of the enzyme@MOF compound is greatly improved, and the enzyme can be rapidly and effectively packaged. However, this method has a certain requirement for the nature of the enzyme-enzymes with negative charges on their surface are more prone to aggregation of metal ions and thus promote the formation of MOF complexes. To solve the problem of the versatility of this method, current research has focused mainly on the destructive complex modification of enzymes to cause metal ions to accumulate around the enzymes, thereby promoting nucleation of crystals. However, the preparation process of the method is complex, and is not beneficial to popularization and application. Therefore, there is a need to develop a general efficient enzyme @ MOF synthesis method that has no special requirements on the enzyme properties. Disclosure of Invention In order to overcome the defects in the prior art, the invention provides a method for efficiently and universally coating enzyme through seed crystal guiding, which solves the problems of the universality, the synthesis efficiency and the difficulty in nondestructively coating enzyme of the existing solution phase enzyme@MOF compound synthesis method (coprecipitation method, biomimetic mineralization method and improved method thereof). In order to achieve the above purpose, the present invention is realized by the following technical scheme: The invention provides a method for guiding a high-efficiency universal synthetase@MOF compound through seed crystals, which is characterized in that in the process of encapsulating the enzyme by utilizing the MOF, the slow nucleation stage is skipped by adding the seed crystals corresponding to the MOF so as to achieve the purpose of rapidly synthesizing the synthetase@MOF compound. Seed crystals are introduced in the synthesis process of the enzyme@MOF, and the seed crystals can serve as crystal nuclei for enzyme attachment and MOF crystal growth, and due to the existence of the seed crystals, the slow nucleation stage in the synthesis process of the MOF crystal is skipped, so that the efficiency of wrapping the enzyme by the MOF is greatly improved, namely the synthesis efficiency of the enzyme@MOF is greatly promoted. The invention can provide a general and efficient enzyme@MOF compound synthesis method. Preferably, the enzyme comprises cytochrome C (Cyt C), hemoglobin (HB), myoglobin (MB). Preferably, the MOF is a ZIF-8 metal organic framework. The invention synthesizes the biological composite material of enzyme @ ZIF-8 through a se