Rigid-Flexible Layered Immobilization Enables Precise Confinement and Dynamic Activation of Small Enzymes.

Area of Science:

• Biotechnology
• Materials Science
• Chemical Engineering

Background:

• Small enzymes (<50 kDa) face challenges in immobilization, including unstable interactions, low activity retention, and lack of specificity.
• Existing immobilization strategies often fail to balance enzyme stability with accessibility for industrial applications.

Purpose of the Study:

• To develop a novel rigid-flexible layered immobilization strategy for small enzymes.
• To enhance enzyme stability, activity retention, and specificity during immobilization.
• To address the trade-off between stability and accessibility in enzyme immobilization systems.

Main Methods:

• Integration of networked metal-organic frameworks (MOFs) into flexible hydrogels to create a dual-pore system.
• Utilizing MOF micropores for enzyme confinement and hydrogel macropores for mass transfer.
• Employing molecular docking to analyze substrate-enzyme interactions and conformational changes.

Main Results:

• The MOF-hydrogel system effectively stabilized enzyme conformation and strengthened enzyme-matrix interactions, preventing leakage.
• The dual-pore system resolved the stability-accessibility trade-off, with MOFs reducing substrate-enzyme distance by 34.2%.
• Immobilized horseradish peroxidase demonstrated robust conversion efficiency over 40 cycles in industrial biocatalysis.
• Immobilized thrombin significantly reduced murine wound bleeding (55%), showing potential for wound hemostasis.

Conclusions:

• The proposed rigid-flexible layered immobilization strategy offers a scalable and effective platform for enhancing enzyme performance.
• This approach holds significant promise for advancing sustainable biocatalysis and developing novel biomedical solutions.
• The synergistic system provides nanoscale precision and macroscale responsiveness for diverse applications.