Low Operational Stability of Enzymes in Dry Organic Solvents: Changes in the Active Site Might Affect Catalysis

Abstract

The potential of enzyme catalysis in organic solvents for synthetic applications has been overshadowed by the fact that their catalytic properties are affected by organic solvents. In addition, it has recently been shown that an enzyme’s initial activity diminishes considerably after prolonged exposure to organic media. Studies geared towards understanding this last drawback have yielded unclear results. In the present work we decided to use electron paramagnetic resonance spectroscopy (EPR) to study the motion of an active site spin label (a nitroxide free radical) during 96 h of exposure of the serine protease subtilisin Carlsberg to four different organic solvents. Our EPR data shows a typical two component spectra that was quantified by the ratio of the anisotropic and isotropic signals. The isotropic component, associated with a mobile nitroxide free radical, increases during prolonged exposure to all solvents used in the study. The maximum increase (of 43%) was observed in 1,4-dioxane. Based on these and previous studies we suggest that prolonged exposure of the enzyme to these solvents provokes a cascade of events that could induce substrates to adopt different binding conformations. This is the first EPR study of the motion of an active-site spin label during prolonged exposure of an enzyme to organic solvents ever reported.

Effect of enzyme dehydration on alcalase‐catalyzed dipeptide synthesis in near‐anhydrous organic media

“…They explain this low storage stability by a possible rearrangement of water molecules within the enzyme or by small perturbations around the active site . A further study proposes that the solvent strips the water molecules and solvent molecules move into the enzyme fold that brings about polarity changes in the active site, which could in turn induce substrates to adopt a different binding conformation . This change of the active site could also well be the case in this study, where the molecular sieves enhance the stripping of the water molecules and the penetration of the solvent molecules into the enzyme.…”

Thermodynamic activity‐based intrinsic enzyme kinetic sheds light on enzyme–solvent interactions

“…Therefore, it has to be assumed that MTBE hinders the intrinsic enzyme catalytic steps and compromises the performance of the enzyme, although the conclusion on distinct solvent effects is difficult in multi‐component reaction systems. Considering the stabilizing effect of MTBE on Lb ADH, however, our data suggest that there might be an enzyme–solvent interaction that bind within the active center or impair the enzyme flexibility that is important for the catalytic activity of alcohol dehydrogenases . Further data obtained under addition of both other substrates and solvents will be required to verify this assumption and to further allow for distinction of individual solvent effects.…”

“…Considering the stabilizing effect of MTBE on LbADH, however, our data suggest that there might be an enzyme-solvent interaction that bind within the active center 12,16,18,40 or impair the enzyme flexibility that is important for the catalytic activity of alcohol dehydrogenases. 15,41,42 Further data obtained under addition of both other substrates and solvents will be required to verify this assumption and to further allow for distinction of individual solvent effects. Still, the concentration-based comparison of the buffer and the buffer/MTBE systems indicate the preferred reaction in presence of MTBE despite these negative enzyme-solvent interactions.…”