Biomolecular Condensation and l -Cysteine Signaling Activate Dormant Protease Activity of Papain Droplets: Application toward Efficient Meat Tenderization

Proteases are an important class of proteolytic enzymes having great importance in both basic science and industrial applications. In this context, the protease activity of papain and its regulation by l-cysteine are poorly understood in the literature. Herein, we discover that papain undergoes biomolecular condensation via spontaneous liquid-liquid phase separation (LLPS) under macromolecular crowding primarily through the involvement of intermolecular hydrophobic interactions. Although native free papain is found to be active toward synthetic and protein substrates, the proteolytic digestion produces heterogeneous peptide aggregates. In contrast, we found that papain droplets remain dormant toward protein digestion owing to the disulfide linkage of the active cysteine residue (Cys-25) in its compact conformational state. Further, we show that the protease activity of phase-separated papain can be reactivated in the presence of l-cysteine to produce uniform soluble peptide fragments. Our findings indicate that although disulfide linkages are not necessary for the phase separation of papain, upon phase separation, intermolecular interactions between phase-separated papain result in the formation of disulfide linkages involving active Cys-25 residues. Finally, we show that the activated papain droplets can remarkably boost the efficacy of meat tenderization compared to free papain. Therefore, the present study not only provides a fundamental understanding of the LLPS of papain and its reactivation by l-cysteine but also illustrates an innovative platform for technological advancement.