Despite these problems, water-organic solvent two-phase systems have been
successfully used to transform highly lipophilic substrates such as steroids [60], fats
[61], and alkenes [62]. In addition, the use of biphasic solvent systems was
beneficial for the asymmetric epoxidation of alkenes (Sect. 2.3.3.3) to minimize
toxic effects of the epoxide produced by Nocardia corallina cells [63].
Enzyme Suspended in a Monophasic Organic Solution
Replacing all of the bulk water (which accounts for >98%) by a water-immiscible
organic solvent leads to a suspension of the solid enzyme in a monophasic organic
solution [64, 65]. Since enzymes are insoluble in organic media, such reactions
resemble a heterogeneous catalytic system. Although the biocatalyst seems to be
‘dry’ on a macroscopic level, it must maintain the necessary residual structural
water to remain catalytically active. Most of the research on such systems (which
have proved to be extremely reliable, versatile and easy to use) has been performed
during the 1980s, but it is striking that the first biotransformation of this kind was
already reported in 1900! [66]. Due to the importance of this technique and its
simplicity, all of the examples discussed below have been performed using solid
‘dry’ enzymes in organic solvents having a water content of <2%. However, it
should be kept in mind that the catalytic activity of enzymes in nonconventional
solvent systems is significantly reduced (often by one order of magnitude) compared to the activity in water [67].
The remarkable catalytic activity of solid proteins in organic solvents can be
explained by their special properties [68]. In contrast to densely packed crystals of
organic compounds of comparatively low molecular weight, which form rather
dense and impenetrable structures, solid proteins represent soft and delicate aggregates. Since the average (monomeric) protein used in biotransformations has a
diameter of ~5 nm (50 Å), there is limited contact between the single protein
molecules thereby allowing minor conformational changes consonant with formation of the enzyme – substrate complex [69]. The total surface of solid enzymes is
within the range of 1–3 Â 10
6 m
2 /kg, which is close to that of silica or activated
carbon. About one to two thirds of the total volume is hollow, with large solventfilled cavities and channels running through a ‘sponge-like’ macroscopic aggregate.
Thus, if sufficient agitation is provided, the substrate is not only transformed by the
active sites exposed to the surface of the crystal but also at those buried inside. In
order to tune a biocatalytic reaction in a monophasic water-immiscible organic
solvent system, the following parameters should be considered [70, 71]:
pH-Memory One particularly important aspect is the effect of the pH of the
reaction medium, which cannot be measured or controlled easily in organic solutions that lack a distinct aqueous phase [72]. However, the pH determines the
ionization state of the enzyme and hence its conformation and its catalytic properties, such as activity and selectivity. Since the ionization state of a protein does not
change when placed in an organic solvent, but remains ‘frozen’, it is important to
employ solid enzymes that have been recovered by lyophilization or precipitation
from a buffer at their pH optimum [73]. The latter fact has vividly been described as
the ‘pH-memory’ of enzymes.
3.1 Enzymes in Organic Solvents
319
successfully used to transform highly lipophilic substrates such as steroids [60], fats
[61], and alkenes [62]. In addition, the use of biphasic solvent systems was
beneficial for the asymmetric epoxidation of alkenes (Sect. 2.3.3.3) to minimize
toxic effects of the epoxide produced by Nocardia corallina cells [63].
Enzyme Suspended in a Monophasic Organic Solution
Replacing all of the bulk water (which accounts for >98%) by a water-immiscible
organic solvent leads to a suspension of the solid enzyme in a monophasic organic
solution [64, 65]. Since enzymes are insoluble in organic media, such reactions
resemble a heterogeneous catalytic system. Although the biocatalyst seems to be
‘dry’ on a macroscopic level, it must maintain the necessary residual structural
water to remain catalytically active. Most of the research on such systems (which
have proved to be extremely reliable, versatile and easy to use) has been performed
during the 1980s, but it is striking that the first biotransformation of this kind was
already reported in 1900! [66]. Due to the importance of this technique and its
simplicity, all of the examples discussed below have been performed using solid
‘dry’ enzymes in organic solvents having a water content of <2%. However, it
should be kept in mind that the catalytic activity of enzymes in nonconventional
solvent systems is significantly reduced (often by one order of magnitude) compared to the activity in water [67].
The remarkable catalytic activity of solid proteins in organic solvents can be
explained by their special properties [68]. In contrast to densely packed crystals of
organic compounds of comparatively low molecular weight, which form rather
dense and impenetrable structures, solid proteins represent soft and delicate aggregates. Since the average (monomeric) protein used in biotransformations has a
diameter of ~5 nm (50 Å), there is limited contact between the single protein
molecules thereby allowing minor conformational changes consonant with formation of the enzyme – substrate complex [69]. The total surface of solid enzymes is
within the range of 1–3 Â 10
6 m
2 /kg, which is close to that of silica or activated
carbon. About one to two thirds of the total volume is hollow, with large solventfilled cavities and channels running through a ‘sponge-like’ macroscopic aggregate.
Thus, if sufficient agitation is provided, the substrate is not only transformed by the
active sites exposed to the surface of the crystal but also at those buried inside. In
order to tune a biocatalytic reaction in a monophasic water-immiscible organic
solvent system, the following parameters should be considered [70, 71]:
pH-Memory One particularly important aspect is the effect of the pH of the
reaction medium, which cannot be measured or controlled easily in organic solutions that lack a distinct aqueous phase [72]. However, the pH determines the
ionization state of the enzyme and hence its conformation and its catalytic properties, such as activity and selectivity. Since the ionization state of a protein does not
change when placed in an organic solvent, but remains ‘frozen’, it is important to
employ solid enzymes that have been recovered by lyophilization or precipitation
from a buffer at their pH optimum [73]. The latter fact has vividly been described as
the ‘pH-memory’ of enzymes.
3.1 Enzymes in Organic Solvents
319