The Role of Glycerol in Preserving Proteins Needs to Be Reconsidered

Abstract

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.

Discovery and mechanism-guided engineering of BHET hydrolases for improved PET recycling and upcycling

“…The computational study, especially molecular dynamics (MD) simulation, can provide a molecular understanding of the identified enzymes 43,[51][52][53][54][55] . Towards in silico characterization of Phase III, the predicted 3D structure of ChryBHETase by AlphaFold2 lacked two regions (residues 251-272 and 245-369) compared to BsEst and TfCa, as shown in sequence alignment (Fig.…”

How Can Deep Eutectic Systems Promote Greener Processes in Medicinal Chemistry and Drug Discovery?

“…It is also important that it should confer stability and maintain the enzyme activity. It has been reported that enzymes can remain stable and active in non-aqueous solvents, such as glycerol, but this is not applicable to all classes of enzymes [ 9 , 10 , 11 ]. Other alternatives have been searched, namely the use of deep eutectic systems (DESs) [ 12 , 13 ].…”

Asymmetric Reduction of Cyclic Imines by Imine Reductase Enzymes in Non‐Conventional Solvents

“…[3] However, recent reports indicate that the stabilizing effect is not universally applicable to all class of enzymes. [4] Various methodologies reporting the use of non-conventional media for biocatalyzed processes have been extensively reviewed. [5] Deep Eutectic Solvents (DESs) are eutectic mixtures commonly obtained by mixing, heating and stirring a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD) in specific molar ratios.…”

“…Among hydrophilic solvents, glycerol can mimic the effect of water and is reported to preserve enzyme structure [3] . However, recent reports indicate that the stabilizing effect is not universally applicable to all class of enzymes [4] . Various methodologies reporting the use of non‐conventional media for biocatalyzed processes have been extensively reviewed [5] .…”