Effects of protein charge and charge patterning on complex coacervation for enzymatic microreactors

Complex coacervation is a liquid-liquid phase separation phenomenon that has shown promise encapsulating proteins and improving protein stability to a variety of perturbations. Cells utilize the process of complex coacervation to create microcompartments, termed membraneless organelles, for increased spatiotemporal control in cellular functions. Here, polypeptide-tagged green fluorescent protein (GFP) mutants and poly(4-vinyl N-methylpyridinium) were used to investigate the effects of protein charge patterning on coacervate phase behavior. Tagged mutants achieved coacervate concentrations higher than previously reported while retaining their secondary structure, suggesting that complex coacervation is a potentially attractive avenue for encapsulating enzymes for biocatalytic applications. Preliminary data has indicated the ability to form liquid-liquid phase separated microcompartments containing multiple enzymes. Using a model colorimetric cascade reaction, these multi-enzyme-polyelectrolyte coacervates have been used to evaluate the catalytic efficiency in biomimetic microcompartments and have the potential to inform the design of novel microcompartments for the production of industrially-relevant chemicals.