The Role of Glycerol in Preserving Proteins Needs to Be Reconsidered

The safe and efficient stabilization and preservation of enzymes, therapeutic proteins, and biomaterials are increasingly important in biology, medicine, and pharmaceutics. Various protectants have been explored, but their protective actions on protein structures remain unclear. Herein, we present an all-around protectant–protein interaction landscape by investigating the behaviors of Bacillus subtilis lipase A (BSLA), cellobiohydrolase I from Trichoderma reesei (CBHI), and endoglucanase from Penicillium Verruculosum (EG) in various concentrations of glycerol. Surprisingly, decreased, neutralization, and activation effects were observed for three industrial enzymes during the long-term storage examination, respectively, demonstrating that a universal condition for protein preservation might not exist. Alignment of the experimental catalytic activity profiles and computational molecular dynamics simulation reveals that (1) the overall structure of enzymes in glycerol remains stable; (2) specific activity reduction mainly results from three factors: (a) increased structural compactness, (b) overall water stripping, and (c) competitive inhibition by glycerol in the substrate binding site; (3) H-bond interactions are the main driving force that governs the structural dynamics, water stripping, and glycerol accumulation in glycerol cosolvents. Also, these gained insights are most likely to be transferred to other polyol additive systems for rationally stabilizing and preserving biomaterials in structural biology, biocatalysis, and biotransformation fields.