Self-Assembled Multimeric-Enzyme Nanoreactor for Robust and Efficient Biocatalysis

Area of Science:

• Biocatalysis
• Synthetic Biology
• Nanotechnology

Background:

• Enzyme cascades are crucial for biocatalysis, but their efficiency is limited by enzyme stability and spatial arrangement.
• Developing stable, reusable multienzyme catalysts remains a significant challenge in enzyme engineering.

Purpose of the Study:

• To create ultrastable multimeric enzyme nanoclusters (MENCs) for enhanced enzyme cascade performance.
• To demonstrate a novel strategy for constructing self-assembled, covalently coupled supramolecular multienzyme nanodevices.

Main Methods:

• Utilized the SpyTag/SpyCatcher system for orthogonal protein ligation.
• Fused SpyCatcher to a dimeric cytochrome P450 monooxygenase mutant (P450BM3m) and SpyTag to a tetrameric glucose dehydrogenase (GDH).
• Investigated the self-assembly, morphology, and catalytic activity of the resulting MENCs.

Main Results:

• Successfully formed stable MENCs with a two-dimensional layerlike nanoscale architecture.
• Achieved efficient NADPH regeneration and indole conversion to indigo.
• Demonstrated a >3-fold increase in the initial rate of indigo synthesis.
• Showcased significant improvements in MENC stability and reusability compared to free enzyme mixtures.

Conclusions:

• The SpyTag/SpyCatcher system provides a versatile and efficient strategy for constructing stable, multifunctional biocatalysts.
• MENCs offer enhanced performance and stability, showing promise for applications in metabolic engineering and synthetic biology.