Pluronic-Based Nanocarrier Platform Encapsulating Two Enzymes for Cascade Reactions.

Enzyme immobilization is very important for diverse enzyme applications. Particularly, there is a growing need for coimmobilization of multiple enzymes for biosensing and synthetic applications. However, it is still challenging to coimmobilize two enzymes with desirable features, including high immobilization yield, retention of enzymatic activity, and low leaching. In this study, we demonstrated that a pluronic-based nanocarrier (PNC) can be an encapsulation platform for immobilization of various single enzymes. Since the PNC is temperature-sensitive, a simple temperature change from 4 to 37 °C led to a substantial size reduction and enzyme encapsulation. All six enzymes tested were encapsulated by the PNC in high yield (∼90%) with the retained enzymatic activity (>95%). The leaching of encapsulated enzymes was very minimal (<0.13% for 2 weeks). Then, we demonstrated that the PNC can efficiently coencapsulate two enzymes, formate dehydrogenase (FDH) and mannitol dehydrogenase (MDH), for a cascade reaction producing d-mannitol. Coencapsulation of FDH and MDH resulted in an over 10-fold increase in d-mannitol production compared to the free mix of FDH and MDH, likely due to the enhanced local concentrations of FDH and MDH inside the PNC.