Non-covalent binding tags for batch and flow biocatalysis

Area of Science:

• Biocatalysis and Enzyme Engineering
• Biotechnology and Protein Chemistry

Background:

• Enzyme immobilization enhances biocatalysis but often requires costly chemical modification of carriers.
• Existing immobilization methods lack specificity and can increase processing costs.

Purpose of the Study:

• To investigate novel enzyme immobilization strategies using specific binding tags on cellulose and silica carriers.
• To evaluate the performance of industrially relevant enzymes (transaminases, imine reductase/glucose oxidoreductase fusion) after immobilization.

Main Methods:

• Fusing known binding tags (Bacillus cereus CotB peptide, Clostridium thermocellum cellulose binding domain) to model and industrial proteins.
• Assessing binding affinity (K_d values) and enzyme activity post-immobilization on respective carriers.
• Testing immobilized enzymes in repetitive batch and continuous-flow reactors.

Main Results:

• Both binding tags demonstrated high-affinity, specific binding to their respective carriers (cellulose and silica).
• The silica-binding peptide caused protein aggregation, while the cellulose-binding domain allowed immobilization but caused activity loss in some enzymes.
• A transaminase-cellulose binding domain fusion was successfully applied in both batch and continuous-flow reactors.

Conclusions:

• Specific binding tags offer a cost-effective alternative to chemical modification for enzyme immobilization.
• The cellulose-binding domain (CBDclos) shows promise for enzyme immobilization in flow chemistry, despite initial activity challenges.
• This study demonstrates the potential of engineered binding tags for simplified and efficient enzyme immobilization in industrial biocatalysis.