cyanohydrins [25], or acyl migration [26] are water-dependent and are therefore
largely suppressed in an organic medium.
• Immobilization of enzymes is not necessary because they may be recovered by
simple filtration after the reaction due to their insolubility in organic solvents.
Nevertheless, if it is desired, experimentally simple adsorption onto the surface
of a cheap macroscopic carrier such as diatomaceous earth (Celite), silica, or
glass beads is possible. Desorption from the carrier into the medium – ‘leaking’ –
is largely impeded in a lipophilic environment.
• Since many of the reactions which are responsible for the denaturation of
enzymes (see Sect. 1.4.1) are hydrolytic reactions and therefore require water,
it can be expected that enzymes should be more stable in an environment of low
water content [27, 28]. For instance, porcine pancreatic lipase is active for many
hours at 100
C in a 99% organic medium but it is rapidly denaturated at this
temperature when placed in pure water [29].
• Due to the conformational change (i.e., a partial unfolding and refolding) of the
enzyme during the formation of the enzyme – substrate complex (the ‘inducedfit’), numerous hydrogen bonds are reversibly broken and reformed. This process is greatly facilitated in an aqueous medium, where the broken bonds are
rapidly replaced by hydrogen bonds to the surrounding water. Thus, it serves as a
‘molecular lubricant’ [22]. In an organic solvent, this process is impeded and, as
a consequence, enzymes appear to be there more ‘rigid’ [30]. Thus, it is often
possible to control some of the enzyme’s catalytic properties such as the
substrate specificity [17, 31–33], the chemo- [34], regio- [35] and
enantioselectivity [36–39] by variation of the solvent (Sect. 3.1.7).
• The most important advantage, however, is the possibility of shifting thermodynamic equilibria to favor synthesis over hydrolysis. Thus, by using hydrolase
enzymes (mainly lipases and proteases), esters [40–42], polyesters [43, 44],
lactones [45, 46], amides [37, 47], and peptides [48] can be synthesized in a
chemo-, regio-, and enantioselective manner.
The solvent systems which have commonly been used for enzyme-catalyzed
reactions containing organic media can be classified into three different categories.
Enzyme Dissolved in a Monophasic Aqueous-Organic Solution
The enzyme, the substrate and/or product are dissolved in a monophasic solution
consisting of water and a water-miscible organic cosolvent, such as dimethyl
sulfoxide, dimethyl formamide, tetrahydrofuran, dioxane, acetone or one of the
lower alcohols, e.g., iso-propanol or tert-butanol. Systems of this type are mainly
used for the transformation of lipophilic substrates, which are sparingly soluble in
an aqueous system alone and which would therefore be impeded by low reaction
rates. In some cases, selectivities of esterases and proteases may be enhanced by using
water-miscible organic cosolvents (see Scheme 2.42). As a rule of thumb, most watermiscible solvents can be applied in concentrations up to ~10–20% of the total volume,
in rare enzyme/solvent combinations even 50–70% of cosolvent are tolerated. If the
proportion of the organic solvent exceeds a certain threshold, the essential structural
water is stripped from the enzyme’s surface leading to deactivation. Only rarely do
3.1 Enzymes in Organic Solvents
317
largely suppressed in an organic medium.
• Immobilization of enzymes is not necessary because they may be recovered by
simple filtration after the reaction due to their insolubility in organic solvents.
Nevertheless, if it is desired, experimentally simple adsorption onto the surface
of a cheap macroscopic carrier such as diatomaceous earth (Celite), silica, or
glass beads is possible. Desorption from the carrier into the medium – ‘leaking’ –
is largely impeded in a lipophilic environment.
• Since many of the reactions which are responsible for the denaturation of
enzymes (see Sect. 1.4.1) are hydrolytic reactions and therefore require water,
it can be expected that enzymes should be more stable in an environment of low
water content [27, 28]. For instance, porcine pancreatic lipase is active for many
hours at 100
C in a 99% organic medium but it is rapidly denaturated at this
temperature when placed in pure water [29].
• Due to the conformational change (i.e., a partial unfolding and refolding) of the
enzyme during the formation of the enzyme – substrate complex (the ‘inducedfit’), numerous hydrogen bonds are reversibly broken and reformed. This process is greatly facilitated in an aqueous medium, where the broken bonds are
rapidly replaced by hydrogen bonds to the surrounding water. Thus, it serves as a
‘molecular lubricant’ [22]. In an organic solvent, this process is impeded and, as
a consequence, enzymes appear to be there more ‘rigid’ [30]. Thus, it is often
possible to control some of the enzyme’s catalytic properties such as the
substrate specificity [17, 31–33], the chemo- [34], regio- [35] and
enantioselectivity [36–39] by variation of the solvent (Sect. 3.1.7).
• The most important advantage, however, is the possibility of shifting thermodynamic equilibria to favor synthesis over hydrolysis. Thus, by using hydrolase
enzymes (mainly lipases and proteases), esters [40–42], polyesters [43, 44],
lactones [45, 46], amides [37, 47], and peptides [48] can be synthesized in a
chemo-, regio-, and enantioselective manner.
The solvent systems which have commonly been used for enzyme-catalyzed
reactions containing organic media can be classified into three different categories.
Enzyme Dissolved in a Monophasic Aqueous-Organic Solution
The enzyme, the substrate and/or product are dissolved in a monophasic solution
consisting of water and a water-miscible organic cosolvent, such as dimethyl
sulfoxide, dimethyl formamide, tetrahydrofuran, dioxane, acetone or one of the
lower alcohols, e.g., iso-propanol or tert-butanol. Systems of this type are mainly
used for the transformation of lipophilic substrates, which are sparingly soluble in
an aqueous system alone and which would therefore be impeded by low reaction
rates. In some cases, selectivities of esterases and proteases may be enhanced by using
water-miscible organic cosolvents (see Scheme 2.42). As a rule of thumb, most watermiscible solvents can be applied in concentrations up to ~10–20% of the total volume,
in rare enzyme/solvent combinations even 50–70% of cosolvent are tolerated. If the
proportion of the organic solvent exceeds a certain threshold, the essential structural
water is stripped from the enzyme’s surface leading to deactivation. Only rarely do
3.1 Enzymes in Organic Solvents
317