Organic solvents were used to either precipitate enzyme or to study denaturation.
The reason why nonaqueous enzymology has generated so much excitement is that enzymes exhibit striking new properties in organic solvents. For example, in organic solvents enzymes often catalyze reactions impossible in water and are very stable.
Furthermore, enzyme selectivity in organic solvents is not only distinct from that in water but can be markedly controlled, and even reversed, by the solvent; this ‘solvent engineering’ thus provides an alternative to protein engineering.
But it was not as easy as it seems, nature has intended enzymes to work better in aqueous solvents than in organic solvents. There are many reasons, if enzymes are not optimally used in organic solvents, their activity reduce remarkably. e.g.
- Diffusional limitations in organic solvents
- Active center blockage
- Conformational change
- Unfavorable energetics of substrate desolvation
- Transition state destabilization
- Reduced conformational mobility
- Suboptimal pH situation
But there is solution of each problem; like if we vigorously agitate enzyme suspension or use small enzyme particles, there will be no diffusional limitation in organic solvents (1).
In the same way for problem (2) use crystalline instead of amorphous enzyme. For (3) Use lyoprotectants; alternatively, prepare enzyme complexes with amphiphiles soluble in organic solvents.
For (4) Select the solvent expected to yield unfavorable solvent-substrate interactions. For (5) Select the solvent expected to yield favorable interactions with the transition state.
For (6) Optimize water activity (aw); hydrate the solvent; use hydrophobic solvents; use water-mimicking and denaturing cosolvent additives. for (7) Dehydrate from aqueous solution of the pH optimal for enzymatic activity; use organic-phase buffers.
As we can see above, there are two possibilities for non-aqueous media:
Low water system: enzymes in nearly anhydrous solvents, reverse micelles
High water system: water miscible organic solvents (co-solvents), organic-aqueous biphasic systems
When/why Organic Solvent is better?
- When substrate(s) have greater solubility in organic solvents
- Shift of reaction equilibria in desirable directions such as use of hydrolases for synthetic reactions
- Reduced risk of microbial growth
- Enhanced thermoslability
- Recovery and reusability of enzyme even without immobilization
- More energy efficient downstream processing when volatile solvents are used
- Convenient to use ‘moisture-sensitive’ substrates/reagents like acid anhydrides
- Possible control of substrate specificity, regiospecificity and enantioselectivity
- Organic synthesis; e.g. peptide synthesis, interesterification of oils and fats etc.
- Analysis and detection e.g. Horseradish peroxidase with a chromogenic substrate
- Oligomerization and polymerization