fluids may react with sensitive groups located at the enzyme’s surface causing a loss
of activity. For instance, carbon dioxide is known to reversibly react with ε-amino
groups of lysine residues by forming carbamates, going in hand with the removal/
formation of a positive charge at the enzyme’s surface [148, 149]. The main use of
supercritical gases as solvents is the production of ‘natural’ compounds used in
cosmetics and food.
The following basic rules should be considered for the application of solid
(‘dry’) enzymes in organic media having a low water content:
• Hydrophobic solvents are more compatible than hydrophilic ones (log P of the
organic solvent should be greater than ~1.5).
• The water layer bound to the enzyme must be maintained; this is accomplished
by using water-saturated organic solvents or, alternatively, via control of the
water activity.
• The ‘micro-pH’ must be that of the pH-optimum of the enzyme in water, a
prerequisite that is fulfilled if the protein was isolated from an aqueous solution
at the pH-optimum.
• Stirring, shaking, or sonication is necessary in order to maximize diffusion of
substrate to the catalyst’s surface.
• The addition of enzyme-stabilizing agents may improve the stability of the solid
enzyme preparation significantly.
3.1.1 Ester Synthesis
Esterification
In every synthetic reaction where a net amount of water is formed (such as an ester
synthesis from an alcohol and a carboxylic acid [40–42]) physicochemical problems arise. Due to the fact that the lipophilic solvent (log P > 1.5) is unable to
accommodate the water which is gradually produced during the course of the
reaction, it is collected at the hydrophilic enzyme surface. As a consequence, the
water forms a discrete aqueous phase which entraps the enzyme, finally separating
substrate and enzyme from each other by a polar interface, which is difficult to
penetrate for lipophilic substrate/product molecules. Thus, the rate slows down and
the reaction may cease before reaching the desired extent of conversion. Furthermore, at elevated water activity the reverse hydrolysis reaction prevails setting a
low ceiling for the conversion. In order to solve this problem, two techniques have
been developed.
• Removal of water from the system [150] (e.g., by evaporation [151], azeotropic
distillation [152]), or via chemical drying [153, 154] via addition of molecular
sieves or water-scavenging inorganic salts [155].
• Alternatively, the formation of water may be avoided by employing an acyltransfer step rather than an esterification reaction.
3.1 Enzymes in Organic Solvents
325
of activity. For instance, carbon dioxide is known to reversibly react with ε-amino
groups of lysine residues by forming carbamates, going in hand with the removal/
formation of a positive charge at the enzyme’s surface [148, 149]. The main use of
supercritical gases as solvents is the production of ‘natural’ compounds used in
cosmetics and food.
The following basic rules should be considered for the application of solid
(‘dry’) enzymes in organic media having a low water content:
• Hydrophobic solvents are more compatible than hydrophilic ones (log P of the
organic solvent should be greater than ~1.5).
• The water layer bound to the enzyme must be maintained; this is accomplished
by using water-saturated organic solvents or, alternatively, via control of the
water activity.
• The ‘micro-pH’ must be that of the pH-optimum of the enzyme in water, a
prerequisite that is fulfilled if the protein was isolated from an aqueous solution
at the pH-optimum.
• Stirring, shaking, or sonication is necessary in order to maximize diffusion of
substrate to the catalyst’s surface.
• The addition of enzyme-stabilizing agents may improve the stability of the solid
enzyme preparation significantly.
3.1.1 Ester Synthesis
Esterification
In every synthetic reaction where a net amount of water is formed (such as an ester
synthesis from an alcohol and a carboxylic acid [40–42]) physicochemical problems arise. Due to the fact that the lipophilic solvent (log P > 1.5) is unable to
accommodate the water which is gradually produced during the course of the
reaction, it is collected at the hydrophilic enzyme surface. As a consequence, the
water forms a discrete aqueous phase which entraps the enzyme, finally separating
substrate and enzyme from each other by a polar interface, which is difficult to
penetrate for lipophilic substrate/product molecules. Thus, the rate slows down and
the reaction may cease before reaching the desired extent of conversion. Furthermore, at elevated water activity the reverse hydrolysis reaction prevails setting a
low ceiling for the conversion. In order to solve this problem, two techniques have
been developed.
• Removal of water from the system [150] (e.g., by evaporation [151], azeotropic
distillation [152]), or via chemical drying [153, 154] via addition of molecular
sieves or water-scavenging inorganic salts [155].
• Alternatively, the formation of water may be avoided by employing an acyltransfer step rather than an esterification reaction.
3.1 Enzymes in Organic Solvents
325