Enhancing Chymotrypsin Activity and Stability of Capillary Immobilized Enzyme Microreactors Using Zeolitic Imidazolate Frameworks as Encapsulation Materials.

Open-tubular immobilized enzyme microreactors (OT-IMERs) are some of the most widely used enzyme reaction devices due to the advantages of simple preparation and fast sample processing. However, the traditional approaches for OT-IMERs preparation had some defects such as limited enzyme loading amount, susceptibility to complex sample interference, and less stability. Here, we report a strategy for the preparation of highly active and stable OT-IMERs, in which the single-stranded DNA-enzyme composites were immobilized in capillaries and then encapsulated in situ in the capillaries via zeolitic imidazolate frameworks (ZIF-L). The phosphate groups of the DNA adjusted the surface potential of the enzyme to negative values, which could attract cations, such as Zn2+, to promote the formation of ZIF-L for enzyme encapsulation. Using chymotrypsin (ChT) as a model enzyme, the prepared ChT@ZIF-L-IMER has higher activity and better affinity than the free enzyme and ChT-IMER. Moreover, the thermal stability, pH stability, and organic solvent stability of ChT@ZIF-L-IMER were much higher than those of free enzyme and ChT-IMER. Furthermore, the activity of ChT@ZIF-L-IMER was much higher than that of ChT-IMER after ten consecutive reactions. To demonstrate the versatility of this preparation method, we replaced ChT with glucose oxidase (GOx). The stability of GOx@ZIF-L-IMER was also experimentally demonstrated to be superior to that of GOx and GOx-IMER. Finally, ChT@ZIF-L-IMER was used for proteolytic digestion analysis. The results showed that ChT@ZIF-L-IMER had a short digestion time and high digestive efficiency compared with the free enzyme. The present study broadened the synthesis method of OT-IMERs, effectively integrating the advantages of metal-organic frameworks and IMER, and the prepared OT-IMERs significantly improved enzyme stability. All of the results indicated that the IMER prepared by this method had a broad application prospect in capillary electrophoresis-based high-performance enzyme analysis.

Enhancing Enzyme Activity Using Hydrophilic Hollow Layered Double Hydroxides as Encapsulation Carriers.

Enzyme immobilization enables the fabrication of flexible and powerful biocatalytic systems that can meet the needs of green and efficient development in various fields. However, restricted electron and mass transfer during enzymatic reactions and disruption of the enzyme structure during encapsulation limit the wide application of the immobilized enzyme systems. Herein, we report an encapsulation strategy based on hollow-shell-layered double hydroxides (LDHs; ZnCo-LDH) for green and nondestructive enzyme immobilization. Benefiting from the protective and enzyme-friendly microenvironment provided by the hydrophilic hollow structure of ZnCo-LDH, the encapsulated enzyme maintains a nearly natural enzyme biostructure and enhanced stability. Notably, mesoporous ZnCo-LDH with excellent electrical properties considerably facilitates electron and mass transport during enzymatic reactions, exhibiting 5.56 times the catalytic efficiency of free enzymes or traditional enzyme encapsulation systems. The current study broadens the family of encapsulated carriers and alleviates the trade-off between enzyme stability and catalytic activity in the encapsulated state, presenting a promising avenue for the industrial application of the enzyme.