Towards a rational understanding of serine protease catalytic activity in organic solvents

Non-aqueous enzymology has become a very active and promising area of research for the synthesis of pharmaceutical products in a more environmentally friendly manner in the past years. The main limitation however, has been that enzyme activity is still much lower in organic solvents than in water.; In this investigation, enzymes in organic solvents were activated by co-lyophilization procedures with crown ethers and cyclodextrins, and the contributing factors for such effects were also identified. The addition of methyl-β-CD (MβCD) prior to lyophilization improved enzyme catalytic properties of serine proteases and lipases in organic solvents. Assessment of enzyme structure in aqueous solution, dried powders, and suspensions of those in organic solvents was carried out by analyzing the amide I-IR spectral region (1700–1600 cm−1). These studies revealed that the co-lyophilization with cyclodextrins and crown ethers reduced lyophilization-induced structural perturbations. Enzyme structure and enantioselectivity correlated. It was observed that the more native the enzyme secondary structure was, the higher the enantioselectivity achieved. Addition of water to MβCD co-lyophilized subtilisin powders was detrimental to enzyme activity. Thermal denaturation experiments were also carried out in 1,4-dioxane to get an insight on enzyme flexibility. The higher the thermal denaturation temperature is, the more rigid is the protein conformation. Co-lyophilization with MβCD increased enzyme flexibility when compared to the dried powder. The crown ethers (18-crown-6, 15-crown-5, and 12-crown-4) also increased enzyme flexibility. Concomitantly, the activity in organic solvents increased. Experiments were also performed to study whether the enzyme activation induced by MβCD and crown ethers caused substrate diffusional limitations. The results showed that this was not the case even though the activation by MβCD and 12-crown-4 was several orders of magnitude. This was due to their ability to reduce enzyme particle sizes as demonstrated by scanning electron microscopy. Lastly, enzyme activity and structure were studied after sequential removal of the additive. Enzyme pretreated with the tested additives followed by 10 washing steps showed to be ca. 30–43-fold more active than the lyophilized powder without additives. This showed that crown ethers and MβCD activate enzymes mainly by locking the active site in a favorable conformation for catalysis.