ammonolysis (using lipases [117], esterases, and proteases), amide/peptide synthesis (using proteases), epoxide hydrolysis (using epoxide hydrolases), and glycoside
synthesis (using glycosidases). Even redox-transformations, such as carbonyl
reduction and sulfoxidation are possible [118–124].
Many disadvantages of ILs, such as toxicity, limited biodegradability and high
cost, can be circumvented by a subgroup of ionic liquids termed ‘deep eutectic
solvents’. They are formed by mixing quaternary ammonium salts, e.g. choline
citrate or acetate, with an uncharged hydrogen-bond donating component, e.g. urea,
isosorbide, glycerol or ethylene glycol. These advanced ILs are biodegradable and
sustain enzyme activity remarkably well despite the presence of high concentrations of denaturing agents, such as urea [125–127]. The first proof-of-concept
concerning the use of deep eutectic solvents in biotransformations only appeared
in 2008 [128]. The reactions studied so far encompass transesterification by lipases
and proteases [129, 130], epoxide hydrolysis by epoxide hydrolases [131] and
protease-catalysed peptide synthesis [132]. The reactions generally exhibited
rates and (enantio)selectivities comparable to or higher than those reported for
conventional organic solvents.
Alternatively, enzyme-catalyzed reactions may be performed in nonconventional
media composed of microemulsions and liquid crystals [133]. The use of these
systems, however, requires a great deal of knowledge of bioprocess engineering for
the separation of the surfactant from substrate(s) and/or product(s).
Supercritical Gases Instead of a lipophilic organic solvent, supercritical gases
such as carbon dioxide,
1 freons (e.g., CHF 3 ), hydrocarbons (ethane, ethene, propane), or inorganic compounds (SF 6 , N 2 O) which exhibit solubility properties
similar to that of a hydrocarbon such as hexane, can be used as solvent or cosolvent
for the enzymatic transformation of lipophilic organic compounds [134–
137]. Enzymes are as stable in these media as in lipophilic organic solvents. The
use of supercritical gases is not restricted to a particular class of enzyme but, not
surprisingly, the use of hydrolases is dominant. For instance, esterification [138],
transesterification [139, 140], alcoholysis [141], and hydrolysis [142] are known as
well as hydroxylation [143] and dehydrogenation reactions [144]. The most striking
advantages of this type of solvent are a lack of toxicity, easy removal and the low
viscosity, which is intermediate between those of gases and those of liquids. This
latter property ensures high diffusion, being about one to two orders of magnitude
higher than in common solvents. Furthermore, small variations in temperature or
pressure may result in large solubility changes near the critical point, which allows
to control an enzyme’s catalytic properties such as reaction rate or stereoselectivity
[145]. However, some disadvantages should be mentioned. The high-pressure
equipment, that must withstand several hundred atmospheres pressure, requires a
considerable initial investment and the depressurization step may cause enzyme
denaturation due to mechanical stress [146, 147]. In addition, some supercritical
1 T crit 31
C and p crit 73 bar.
324
3 Special Techniques
synthesis (using glycosidases). Even redox-transformations, such as carbonyl
reduction and sulfoxidation are possible [118–124].
Many disadvantages of ILs, such as toxicity, limited biodegradability and high
cost, can be circumvented by a subgroup of ionic liquids termed ‘deep eutectic
solvents’. They are formed by mixing quaternary ammonium salts, e.g. choline
citrate or acetate, with an uncharged hydrogen-bond donating component, e.g. urea,
isosorbide, glycerol or ethylene glycol. These advanced ILs are biodegradable and
sustain enzyme activity remarkably well despite the presence of high concentrations of denaturing agents, such as urea [125–127]. The first proof-of-concept
concerning the use of deep eutectic solvents in biotransformations only appeared
in 2008 [128]. The reactions studied so far encompass transesterification by lipases
and proteases [129, 130], epoxide hydrolysis by epoxide hydrolases [131] and
protease-catalysed peptide synthesis [132]. The reactions generally exhibited
rates and (enantio)selectivities comparable to or higher than those reported for
conventional organic solvents.
Alternatively, enzyme-catalyzed reactions may be performed in nonconventional
media composed of microemulsions and liquid crystals [133]. The use of these
systems, however, requires a great deal of knowledge of bioprocess engineering for
the separation of the surfactant from substrate(s) and/or product(s).
Supercritical Gases Instead of a lipophilic organic solvent, supercritical gases
such as carbon dioxide,
1 freons (e.g., CHF 3 ), hydrocarbons (ethane, ethene, propane), or inorganic compounds (SF 6 , N 2 O) which exhibit solubility properties
similar to that of a hydrocarbon such as hexane, can be used as solvent or cosolvent
for the enzymatic transformation of lipophilic organic compounds [134–
137]. Enzymes are as stable in these media as in lipophilic organic solvents. The
use of supercritical gases is not restricted to a particular class of enzyme but, not
surprisingly, the use of hydrolases is dominant. For instance, esterification [138],
transesterification [139, 140], alcoholysis [141], and hydrolysis [142] are known as
well as hydroxylation [143] and dehydrogenation reactions [144]. The most striking
advantages of this type of solvent are a lack of toxicity, easy removal and the low
viscosity, which is intermediate between those of gases and those of liquids. This
latter property ensures high diffusion, being about one to two orders of magnitude
higher than in common solvents. Furthermore, small variations in temperature or
pressure may result in large solubility changes near the critical point, which allows
to control an enzyme’s catalytic properties such as reaction rate or stereoselectivity
[145]. However, some disadvantages should be mentioned. The high-pressure
equipment, that must withstand several hundred atmospheres pressure, requires a
considerable initial investment and the depressurization step may cause enzyme
denaturation due to mechanical stress [146, 147]. In addition, some supercritical
1 T crit 31
C and p crit 73 bar.
324
3 Special Techniques