Proteins Organic solvents were used to precipitate the enzyme or to study denaturation.
The reason nonaqueous enzymology has generated so much excitement is that enzymes exhibit surprising new properties in organic solvents. For example, in organic solvents enzymes often catalyze impossible reactions in water and are very stable.
Furthermore, the enzymatic selectivity in organic solvents is not only different from that of water, but the solvent can significantly control and even reverse it; This "solvent engineering" provides an alternative to protein engineering.
But it was not as easy as it seems, nature has wanted enzymes to work better in aqueous solvents than in organic solvents. There are many reasons, if enzymes are not used optimally in organic solvents, their activity is markedly reduced. e.g.
Diffusion limitations in organic solvents
Active center lock
Conformational change
Unfavorable energetics of substrate desolvation
Destabilization of the transition state
Reduced conformational mobility
Sub-optimal pH situation
But there is a solution for every problem; as if we vigorously shake the enzyme suspension or use small enzyme particles, there will be no diffusion limitation in organic solvents (1).
In the same way, for problem (2) use crystalline enzyme instead of amorphous. For (3) Use lyoprotectants; alternatively, prepare enzyme complexes with organic solvent soluble amphiphiles.
For (4) Select the solvent that is expected to produce unfavorable substrate-solvent interactions. For (5) Select the solvent that is expected to produce favorable interactions with the transition state.
To (6) Optimize water activity (aw); hydrate the solvent; use hydrophobic solvents; use denaturing co-solvent additives that mimic water. to (7) dehydrate from an aqueous solution of optimum pH for enzymatic activity; use organic phase buffers.
As we can see above, there are two possibilities for non-aqueous media:
Low water content system: enzymes in near anhydrous solvents, reverse micelles
High water content system: water-miscible organic solvents (cosolvents), organic-aqueous biphasic systems
When / why is organic solvent better?
When the substrate (s) have higher solubility in organic solvents
Shifting reaction equilibria in desirable directions, such as using hydrolases for synthetic reactions.
Reduced risk of microbial growth
Improved thermolability
Recovery and reuse of the enzyme even without immobilization.
More energy efficient post-processing when volatile solvents are used
Suitable for using "moisture sensitive" substrates / reagents such as acid anhydrides
Possible control of the specificity, regiospecificity and enantioselectivity of the substrate
Applications:
Organic synthesis; eg synthesis of peptides, interesterification of oils and fats, etc.
Lipolysis
Analysis and detection, p. Ex. Horseradish peroxidase with chromogenic substrate
Oligomerization and polymerization