Combining Protein Phase Separation and Bio-orthogonal Linking to Coimmobilize Enzymes for Cascade Biocatalysis

Area of Science:

• Biocatalysis and Enzyme Engineering
• Materials Science and Nanotechnology
• Synthetic Chemistry

Background:

• Co-immobilization of multiple enzymes for sequential reactions (vectorial biocatalysis) is complex.
• Existing methods for enzyme encapsulation in frameworks like ZIF-8 often lack spatial organization and efficient substrate channeling.

Purpose of the Study:

• To develop a method for ordered co-immobilization of enzymes within ZIF-8 for enhanced biocatalysis.
• To improve yield and enantiomeric excess in enzymatic synthesis through spatial organization and efficient channeling.

Main Methods:

• Utilized protein phase separation induced by Zn2+ ions and bioorthogonal linking (SpyTag/SpyCatcher) for enzyme co-immobilization.
• Engineered aldoketoreductase (AKR) and alcohol dehydrogenase (ADH) with specific peptide fusions.
• Encapsulated engineered enzymes in zeolitic imidazole framework-8 (ZIF-8) via in situ droplet formation.

Main Results:

• Achieved stable, spatially organized co-immobilization of AKR and ADH within ZIF-8 (AAE@ZIF).
• Demonstrated significantly higher yield (91% after 6 cycles) and enantiomeric excess (99.99% ee) compared to traditional encapsulation (72.9% yield).
• Observed enhanced channeling effects due to the ordered enzyme arrangement.

Conclusions:

• Protein phase separation combined with bioorthogonal linking provides an effective strategy for creating organized multi-enzyme systems in ZIF-8.
• This approach offers a robust platform for advanced biocatalysis with improved efficiency and product purity.
• The developed method overcomes limitations of traditional enzyme immobilization techniques.