The role of the reaction medium in lipase-catalyzed esterifications and transesterifications

Article abstract of DOI:10.1016/S0009-3084(98)00040-1

The nature of the reaction medium (different organic solvents and supercritical fluids) markedly influenced the microbial lipase activity in esterification and transesterification reactions employing as substrates natural and synthetic compounds in terms of reaction rates and lipase enantioselectivity. The experimental data obtained show that while there are no substantial correlations between enantioselectivity and some physicochemical characteristics of the solvent as hydrophobicity and dielectric constant, the solvent polarity and hydrophobicity is able to modulate greatly lipase activity. The possible effects of the solvent characteristics on the catalytic performance of the enzymes are discussed and a rationale is proposed to explain the results obtained.

Full text of DOI:10.1016/S0009-3084(98)00040-1

Chemistry and Physics of Lipids
93 (1998) 157–168
The role of the reaction medium in lipase-catalyzed
esterifications and transesterifications
E. Cernia *, C. Palocci, S. Soro
Department of Chemistry, Uni6ersity of Rome ‘La Sapienza’, Piazalle Aldo Moro 5, Rome 00185, Italy
Received 15 December 1997; accepted 30 January 1998
Abstract
The nature of the reaction medium (different organic solvents and supercritical fluids) markedly influenced the
microbial lipase activity in esterification and transesterification reactions employing as substrates natural and
synthetic compounds in terms of reaction rates and lipase enantioselectivity. The experimental data obtained show
that while there are no substantial correlations between enantioselectivity and some physicochemical characteristics of
the solvent as hydrophobicity and dielectric constant, the solvent polarity and hydrophobicity is able to modulate
greatly lipase activity. The possible effects of the solvent characteristics on the catalytic performance of the enzymes
are discussed and a rationale is proposed to explain the results obtained. © 1998 Elsevier Science Ireland Ltd. All
rights reserved.
Keywords: Lipase; Biocatalysis; Organic solvent; Supercritical fluids
1. Introduction
1989; Wescott and Klibanov, 1994). Moreover,
the demand by health authorities for enantiomeri-
cally pure, pharmaceutical active compounds, has
led to the increased use of biocatalysts and in-
creased research in order to obtain enzymes with
higher activity and selectivity (Roberts et al.,
1993).
In this field the lipases are especially widespread
as biocatalysts for selective acylation and deacyla-
tion of natural and synthetic compounds and for
resolution of racemic alcohols and acids
(Bjorkling et al., 1991).
The use of organic media for enzymatic reac-
tions is now well documented and several systems
(biphasic systems, reverse micelles and monopha-
sic systems) and enzymes (oxidoreductases, hydro-
lases and isomerases) have been successfully
employed for synthetic purposes (Chen and Sih,
* Corresponding author. Tel.: +39 6 4469871; fax: +39 6
49913340; e-mail: cernia@axrma.uniromal.it
0009-3084/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved.
PII S0009-3084(98)00040-1

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E. Cernia et al. / Chemistry and Physics of Lipids 93 (1998) 157–168
Table 1
Besides the activity in the field of new and more
Physical properties of supercritical fluids
economical lipolytic enzymes from different
sources (microbial, mammalian and plant
sources), as well as in the field of recombinant
lipases preparation through protein and genetic
engineering, many efforts have been devoted to
optimize reaction conditions in order to improve
lipase performance. A study is being carried out
on the reaction medium in order to modulate the
yield as well as the stereoselectivity and regioselec-
tivity of the biocatalysed reactions (Van Tol et al.,
1995).
Our research group has been involved in the
study of microbial lipases (fungal and bacterial
source) to be employed in biotransformations of
natural and synthetic compounds. On this basis
we have carried out studies on the different
parameters that can be employed to modulate
microbial lipases catalytic activity. Recently we
have devoted a large part of our study to the
investigation of the importance of reaction
medium, employing organic solvents and fluids in
supercritical conditions.
Gas Supercritical fluid
Liquid
Density (g/cm3)
Diffusion (cm2/s)
Viscosity (g/cm*s)
10−3
0.1–1
1
10−1 10−3–10−4
10−4 10−3–10−4
B10−5
10−2
Gas like: High diffusivity (mass transport in complex ma-
trices); low viscosity (favorable flow characteristics).
Liquid like: high solvatating power (dependent on density).
All phenyl ethanol derivatives, acylating agents,
menthol and the primary alcohols (ethanol, bu-
tanol, hexanol, octanol, decyl alcohol, dodecanol,
tetradenanol, 3 - methyl 2 - buten 1 - ol, crotylic
alcohol) were purchased from Aldrich Chemical.
(9) - endo - bicicyclo (2.2.1)hept - 5 - en - 2 - ol was
The use of microbial lipases in non conven-
tional media such as supercritical fluids (SCFs),
has been proposed as a means of improving the
activity and utility of such enzymes in anhydrous
environments (Cernia and Palocci, 1997). It is well
known, that by exploiting the unique solvent
properties of SCFs (Table 1) (viscosity wide varia-
tions are possible with small changes in pressure
and/or temperature) it may be possible to enhance
reactions rates while maintaining or improving
selectivity (Catoni et al., 1996). Separation of
products from reactants can be greatly facilitated
by the ease with which the solvent power of the
SCFs can be adjusted.
2. Materials and methods
2.1. Chemicals
The lipase from Pseudomonas cepacea (Pseu-
domonas SP) was provided by Amano P, and was
immobilised on ACR-silica gel (Catoni et al.,
1996).
Scheme 1. Secondary aliphatic and aromatic alcohols with
different substitution moiety.

E. Cernia et al. / Chemistry and Physics of Lipids 93 (1998) 157–168
159
Fig. 1. General scheme of enzymatic esterification of secondary alcohols in different organic solvents and supercritical carbon
dioxide.
Fig. 2. Esterification reactions catalysed by Pseudomonas cepacea lipase in organic solvents and supercritical carbon dioxide. As
substrates: acetic anhydride and: (A) 1-phenylethanol; (B) 1-(4-chlorophenyl)-ethanol; (C) 1-(4-tert-buthylphenyl)-ethanol. Conver-
sion (%) versus reaction time.

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E. Cernia et al. / Chemistry and Physics of Lipids 93 (1998) 157–168
Table 2
Enantiomeric excess (%) at 360 min and 40°C of esterification reaction of different secondary alcohols and acetic anhydride
biocatalysed by immobilised Pseudomonas cepacea lipase in organic solvents and in supercritical CO2
Substrate
SC–CO2
Hexane
CCl4
Toluene
Benzene
CHCl3
Net3
CH2Cl2
1-Phenyl-ethanol
97
96
80
96
100
99
100
90
42
69
58
88
60
93
71
76
10
88
76
69
70
49
62
13
75
13
88
80
70
89
88
70
100
75
54
87
82
67
73
86
75
100
60
14
93
85
90
80
55
80
100
60
7
67
7
12
4
20
3
4
85
75
85
84
39
78
31
43
16
1-(4-Fluorophenyl)-ethanol
1-(4-Chlorophenyl)-ethanol
1-(4-Bromophenyl)-ethanol
1-(4-Methoxyphenyl)-ethanol
1-(4-tert-Buthylphenyl)-ethanol
1-(2-Bromophenyl)-ethanol
(2,4-Dichlorophenyl)-ethanol
1-(2-Methoxyphenyl)-ethanol
88
1
95
synthesised according to Oberhauser (Ober-
hauser et al., 1987) and Honda (Honda et al.,
1981). (2R*,3S*) Methyl trans 3-(4-meth-
oxyphenyl) glycidate was a generous gift from
Lusochimica (Italy). All solvents were from Carlo
Erba Analyticals, with a maximum water content
of 0.02%.
2.2. Reactions in organic sol6ents
Esterification and transesterification reactions
were carried out in 1.75 ml of organic solvent
containing 0.9 mmol of each alcohol (phenyl
ethanol derivatives menthol and (9)-endo-bici-
cyclo (2.2.1)hept-5-en-2-ol), 0.9 mmol of acylat-
ing agent and from 25 to 60 mg of free and
immobilised Pseudomonas cepacea lipase. Trans-
esterifications reactions were carried out in 3.2
ml of organic solvent containing 9 mmol of
methyl trans 3-(4-methoxyphenyl) glycidate, 9
mmol of each alcohol and 53 mg of free Pseu-
domonas cepacea lipase.
All the reaction mixtures were incubated at
40°C under magnetic stirring at 600 rpm. Sam-
ples were taken for analysis at regular intervals.
2.3. Reaction in supercritical carbon dioxide
The apparatus employed to carry out the re-
actions in supercritical medium (SFE 30 Fison
Instruments) were specially designed to investi-
gate various enzymatic reactions in supercritical
carbon dioxide in a reactor with a reaction cell
volume of 0.9 ml. The pressure was controlled
by a syringe pump that ensured a rapid target
pressure achievement. A 0.35 mmol quantity of
alcohol, 0.35 mmol of acylating agent and 10
mg of immobilised enzyme were introduced to
the reactor. After sealing, pressurisation is
achieved by pumping liquid carbon dioxide to the
Fig. 3. Esterification reactions of 1-phenylethanol and acetic
anhydride catalysed by Pseudomonas cepacea lipase in organic
solvents. Conversion (%) versus solvent characteristics ex-
pressed as log Pow
.

E. Cernia et al. / Chemistry and Physics of Lipids 93 (1998) 157–168
161
Fig. 4. Substrates employed in transesterification reactions catalysed by Pseudomonas cepacea lipase.
Table 3
Conversion (%) and enantiomeric excess (%) at 48 h and 40°C
of transesterification reaction of (9)-norbornenol and vinyl
acetate biocatalysed by immobilised Pseudomonas cepacea li-
pase in organic solvents
Reaction media
Initial rate (vmol/
C (%) e.e. (%)
min)
n-Hexane
Cyclohexane
CCl4
Toluene
Benzene
CHCl3
NEt3
Cyclohexanone
CH2Cl2
THF
Vinyl acetate
Aceton
CH3CN
DMF
Dioxane
0.59
0.80
0.58
0.32
0.26
0.12
0.40
0.38
0.11
0.19
0.33
0.17
0.11
—
48
75
50
49
46
18
52
32
17
32
45
28
20
0
80
65
78
79
84
89
70
82
84
87
72
83
75
—
82
Fig. 5. Scheme of oxidation reaction to obtain (−)-(1S,4S)-bi-
0.19
36
cyclo-(2.2.1)hept-5-en-2-one
(2.2.1)hept-5-en-2-one.
from
(9)-endo-bicyclo-

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E. Cernia et al. / Chemistry and Physics of Lipids 93 (1998) 157–168
Fig. 6. Scheme of transesterification reaction of (9)-endo-bicyclo-(2.2.1)hept-5-en-2-one catalysed by Pseudomonas cepacea lipase
in organic solvents.
desired final pressure (20 MPa), and the reac-
tor is thermostated at 40°C. During the reaction
time a 6-way valve (Rheodyne 7125) allows sam-
ple withdrawal for analysis without depressurisa-
tion. At the end of reaction, depressurisation is
achieved by opening a needle valve above the
reaction cell.
2.4. Chromatographic analysis
Esters production of phenyl ethanol derivatives,
menthol and (9)-endo-bicicyclo (2.2.1)hept-5-en-
2-ol, was followed by gas chromatography (Carlo
Erba 5300) and flame ionisation detector, with
nitrogen as carrier gas at a pressure of 1 bar using
capillary columns. Phenyl ethanol derivatives
were analysed using a polyphenyl-methylsiloxane
column (OV1: 25 m×0.53 mm ID); the enan-
tiomeric resolution was obtained employing two
serial capillary column: the first one achiral (OV1:
10 m×0.53 mm ID or Carbowax: 20 m×0.25
mm ID) the other one chiral (Cp-cyclodextri-B-2,
3,6-M-19: 25 m×0.25 mm ID). Menthol was
detected using a CDX-B column (Cp-cyclodextri-
B-2, 3,6-M-19: 25 m×0.25 mm ID). Progress of
the reaction of -endo-bicicyclo (2.2.1)hept-5-en-2-
ol was followed on OV-1 column (OV 1:10 m×
0.53 mm ID) while enantiomeric excess was
detected on CDX-B column (Cp-cyclodextri-B-2,
3,6-M-19: 25 m×0.25 mm ID). Progress of the
reactions of methyl trans 3-(4-methoxyphenyl)
glycidate and the enantiomeric excess (e.e.) (A.
Gentile, 1992) were followed by HPLC (HPLC
Fig. 7. Transesterification reactions of (9)-endo-bicyclo-
Perkin Elmer Series 10), using a chiral column
(2.2.1)hept-5-en-2-one and vinyl acetate catalysed by Pseu-
(CHIRALCEL OD-H 25×0.46 cm I.D. from
domonas cepacea lipase in organic solvents. Conversion (%)
DAICEL) with a mixture of n-hexane/iso-propyl
versus solvent characteristics expressed as: (A) log Pow; (B)
dielectric constant.
alcohol (90:10) as an eluent phase.

E. Cernia et al. / Chemistry and Physics of Lipids 93 (1998) 157–168
163
Fig. 8. Scheme of transesterification reactions of (9)menthol and vinyl acetate catalysed by Pseudomonas cepacea lipase in organic
solvents.
3. Results and discussion
tions, dramatically depend on the employed solvent
while the use of differently activated substrates do
not substantially improve the reaction rates in the
employed conditions.
In order to explain this dependence we attempted
to correlate the enzymatic activity, with some
physic-chemical properties of the solvent.
The enantiomeric excess values plotted against
solvent dielectric constant (m), the Hildebrand sol-
ubility parameter (l) and log Pow (Log Pow is
defined as the logarithm of the partition coefficient
of a given compound in the standard octanol-water
two-phase system) do not furnish significant re-
sults; otherwise a linear correlation between the
percentage of conversion and log Pow was found
(Fig. 3). The results clearly prove that there is an
increase of lipase activity with increasing solvent
hydrophobicity expressed in terms of log Pow val-
ues.
As model substrates, secondary aliphatic and
aromatic alcohols with different substitution moi-
eties (Scheme 1) were firstly employed in esterifica-
tion reactions with acetic anhydride in some
organic solvents and supercritical carbon dioxide
using as catalyst an immobilized lipase from Pseu-
domonas cepacia.
Fig. 1 shows the reaction scheme of the esterifi-
cation reactions of some of the above mentioned
substrates with acetic anhydride in supercritical
carbon dioxide and organic solvents. Fig. 2 shows
the kinetic curves of some employed substrates and
Table 2 summarizes the enantiomeric excess values
obtained.
As can be clearly seen the percentage of conver-
sion and the enantioselectivity of the studied reac-
Table 4
Conversion (%) and enantiomeric excess (%) at 48 h and 40°C
of transesterification reaction of (9)-menthol and vinyl ac-
etate biocatalysed by immobilised Pseudomonas cepacea lipase
in organic solvents
As far as the results obtained in supercritical
fluids, we observed an higher extent of conversion
in SCCO2 compared to that in organic solvents.
Although the lack of data regarding the dielectric
constant and the log Pow value for supercritical
CO2, the solvent hydrophobicity does not seem to
play an important role in reaction rate enance-
ments. Our results can be explained in terms of
enhanced transport properties, high diffusivity of
solute in supercritical medium as in terms of high
enzyme stability in supercritical phases. Moreover
the use of a reaction medium in a supecritical state
determined a sharp increase of the reaction enan-
tioselectivity with e.e. values (]98%) higher than
those obtained in organic solvents.
Reaction media
C (%)
e.e. (%)
n-Hexane
Cyclohexane
CCl4
Toluene
Benzene
CHCl3
NEt3
CH2Cl2
THF
Aceton
Dodecane
Decane
Dioxane
39
35
26
25
27
12
15
16
19
17
35
34
17
95
96
98
95
94
—
94
94
94
93
95
95
97
Lipases from Pseudomonas cepacia were also
used in the enantioselective transterification reac-
tions using racemic alcohols ((9)-endo-bicyclo

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E. Cernia et al. / Chemistry and Physics of Lipids 93 (1998) 157–168
reported in Fig. 4.
The chiral alcohol (9)-endo-bicyclo (2.2.1)hept-
5-en-2-ol is a useful starting material for the
stereospecific synthesis of cyclopentane systems
such as methaneprostacyclins, nucleoside ana-
logues and Brefeldin A. (Betina et al., 1965; Bartlett
and Greene, 1978; Baudouy et al., 1977; Eichberger
et al., 1986).
Brefeldin A is a fungal metabolite with a chemical
structure containing a macrocyclic lactone ring
which has a wide range of biological activities
(Betina, 1969; Harri et al., 1971). One of the
synthetic pathways to synthesize Brefeldin A em-
ploys a (1S,4S) bicyclopenthanone (Corey and
Wollenberg, 1976; Berger and Faber, 1991).
Therefore we thought it would be interesting to
investigate the lipase catalyzed resolution of the
racemic mixture of (9)-norbornenol from which
optically active chetone can be easily obtained (Fig.
5).
The transesterification reactions were carried out
employing vinyl acetate as acyl donor in different
reaction media (Fig. 6). Besides studies on the use
of different acetyl donors, with different chemical
structures (esters of saturated, insaturated alcohols
and enol acetates), our interest was focused on the
possibility of improving the reaction yield and the
enantioselectivity of the considered reactions using
different organic solvents with different physic-
chemical properties. The percentage of conversion
at fixed reaction time, depends greatly on the
solvent employed but there is no effect on the
enantioselectivity of the process (Table 3). More-
over the e.e. (]80%) values are substantially
independent of the acetyl donor structure.
Fig. 7 shows the plot of the initial velocities in
different reaction media against some physic-chem-
ical properties of the chosen solvents.
Results pointed to the impossibility of finding a
direct correlation with a single parameter.
Menthol, a secondary alcohol of terpenic struc-
ture obtained from Mentha piperita or Mentha
ar6ensis plants by steam distillation (Eccles, 1994)
has three asymmetric carbon atoms in its cyclohex-
ane ring (Bauer et al., 1990; Eccles et al., 1990). Only
the (−)menthol has an effect on the cold receptors
of the human skin (interfere with calcium ions
crossing through the cell membrane causing oscil-
lation in the membrane potential).
Fig. 9. Transesterification reactions of (9)menthol and vinyl
acetate catalysed by Pseudomonas cepacea lipase in organic
solvents. Conversion (%) versus solvent characteristics ex-
pressed as: (A) log Pow; (B) Hildebrand solubility parameter.
(2.2.1)hept-5-en-2-ol, (9) menthol) and esters
(2R*,3S*) methyl trans 3-(4-methoxyphenyl) glyci-
date) as substrates of pharmaceutical interest as

E. Cernia et al. / Chemistry and Physics of Lipids 93 (1998) 157–168
165
Fig. 10. Scheme of transesterification reactions of methyl trans-3-(4-methoxyphenyl)glycidate and buthanol catalysed by Pseu-
domonas cepacea lipase in organic solvents.
Fig. 11. Transesterification reaction of trans-3-(4-
Fig. 12. Transesterification reactions of trans-3-(4-
methoxyphenyl)glycidate and buthanol catalysed by Pseu-
methoxyphenyl)glycidate and buthanol catalysed by Pseu-
domonas cepacea lipase in n-hexane. Conversion (%) and
domonas cepacea lipase in organic solvents. Conversion (%)
enantiomeric excess (%) versus reaction time.
versus reaction time.

166
E. Cernia et al. / Chemistry and Physics of Lipids 93 (1998) 157–168
Fig. 13. Transesterification reactions of trans-3-(4-methoxyphenyl)glycidate and buthanol catalysed by Pseudomonas cepacea lipase
in organic solvents. Conversion (%) versus solvent characteristics expressed as: (A) dielectric constant; (B) log Pow
.
We carried out a study on the possibility of
separating menthol enantiomers using a transter-
ification reaction catalyzed by lipase from Pseu-
domonas cepacia in organic solvents (Fig. 8).
Besides the temperature optimization of the reac-
tion conditions, the substrates molar ratio (men-
thol and vinyl acetate), the amount of the enzyme
used, particular attention was devoted to the pos-
sibility of modulating enzyme activity and selec-
tivity by varying the reaction medium.
The percent of conversion depends greatly on
the solvent employed, the yield varying from 10 to
50% after 96 h reaction time. The e.e. values
(Table 4) are very high (]90%) and independent
from the solvent chosen.
The correlation between enzyme activity and
some physic-chemical properties, such as the sol-
vent dielectric constant, log Pow and the Hylde-
brand solubility parameter, gave us very
interesting results. Only the solvent hydrophobic-
ity and the Hyldebrand solubility parameter (Fig.
9) was an unequivocal correlation found, showing
an enzymatic yield inversely dependent on solvent
polarity.
Diltiazem (1), (+) cis-(2S,3S)-3-acetoxy-5-[2-
(dimethylamino) ethyl] 2,3-dihydro-2-(4-
methoxyphenyl) 1,5-benzothiazepin-4-(5H), is a
very strong vasodilating agent discovered by Tan-
abe Seiyaku, which features calcium channel
blocking activity (Schwartz et al., 1992). Among
the several synthetic approaches that have been
described (Watson et al., 1990; Miyata et al.,
1991) one of the most important is based on the
Tanabe synthesis (Tanabe, 1984) which begins
with the Darzens’ condensation of 4-methoxyben-
zaldehyde (a) with methyl chloroacetate. The sub-
sequent reaction opens the oxirane ring of the
methyl trans 3-(4-methoxyphenyl) glycidate (b)
with 2-nitrothiophenol. Several subsequent steps
(intramolecular ring closure, reduction of the NO2
group, ester hydrolysis, acetylation and N-alkyla-
tion) complete the synthesis. Only the (2R,3S)
methyl trans 3-(4-methoxyphenyl) glycidate is nec-
essary in the synthesis of Diltiazem, however,
after Darzens’ condensation a racemic mixture is
obtained.
Some authors (Kaverna and Sundholm, 1993)

E. Cernia et al. / Chemistry and Physics of Lipids 93 (1998) 157–168
167
4. Conclusions
studied the transesterification reaction of
(2R*,3S*) methyl trans 3-(4-methoxyphenyl) gly-
cidate with aliphatic alcohols catalyzed by Can-
dida cylindracea lipase. They focused their
attention on the water content of different organic
solvents used as reaction media. As a matter of
fact their results show a rough correlation be-
tween the yield of the reaction and the amount of
the water stripped by the organic solvent.
On this basis we decided to study in depth the
influence of the reaction medium in the transester-
ification reaction of (2R*,3S*) methyl trans 3-(4-
methoxyphenyl) glycidate with aliphatic alcohols
employing a lipase from Pseudomonas cepacia
(Fig. 10).
The possibility of varying the activity and the
selectivity of the lipase biocatalysed reactions, by
changing the nature of the reaction medium, can
be considered a useful tool to modulate the chem-
ical interactions between enzyme, substrate and
reaction media with different physico-chemical
properties (Secundo et al., 1992). These studies
not only improve the understanding of enzymes
mechanism of action but even contribute to elabo-
rate better strategies for industrial applications of
lipolytic enzymes.
From the results obtained, carrying out esterifi-
cation or transesterification reactions with model
substrates in different organic solvent, Pseu-
domonas cepacia lipase activity shows a depen-
dence on the physic-chemical properties of the
reaction medium employed. In particular solvent
As reported in Fig. 11 the enzymatic reaction
proceeds very slowly and although the final con-
version approaches 80%, the enantiomeric excess,
calculated for the (2R,3S) enantiomer, decreases
from 55 to 23%.
hydrophobicity, expressed as log Pow
, is the
physicochemical characteristic best correlated
with the enzymatic activity. As matter of fact
apolar and hydrophobic solvent with low dielec-
tric constant values increase the reaction rate,
however, polar solvent or containing heteroatoms,
carbonilyc or amidic groups decrease the reaction
rate. The use of hydrophobic solvents in lipases
biocatalysed reactions preserves enzyme confor-
mational rigidity and stability (even in the pres-
ence of fluids in supercritical conditions).
Moreover the motions in proteins are partially
governed by electrostatic interactions (Affleck et
al., 1992). In fact electrostatic forces are the dom-
inant factor correlating protein structure and
function. Attenuation of such forces by the sol-
vent environment can be dramatic (use of solvents
with high dielectric constant values) particularly
in solvent-accessible regions of enzyme proteins.
On this basis, we could speculate that one or
more molecules of solvents could interact with the
enzyme in or near the substrate binding site and
depending on their physicochemical properties
they could interfere with the transformation of the
substrate, thus affecting the reaction rate. There-
fore according to our hypothesis it could be the
solvent-enzyme complex that influences the activ-
ity. The formation of a such complex requires that
In the condition we employed, the best relation-
ship between yield and enantiomeric excess was
observed within only 48 h reaction times.
The kinetic curves of the transesterification re-
action between butanol and methyl trans 3-(4-
methoxyphenyl) glycidate in different organic
solvents are shown in Fig. 12. As can be seen
clearly the conversion greatly depends on the
reaction medium employed.
In order to better understand these results trials
were carried out to correlate the enzymatic activ-
ity with some physic-chemical characteristics of
the organic solvents. The dielectric constant (m),
Hildebrand solubility parameter (l), solvent den-
sity (z) and solvent hydrophobicity, expressed as
log Pow, were taken into consideration.
In Fig. 13 the dielectric constant and the
log Pow values against the percentage of conver-
sion (after 48 h) are plotted respectively. Solvents
with high solubility parameter and relatively high
dielectric constant values show a common feature
in terms of solvent hydrophobicity showing low
log Pow values. The enzymatic reactions carried
out in these solvents show very low conversion
rates (510%). In contrast hydrophobic solvents
with low solubility parameter and dielectric con-
stant values furnished yields greater than 30%.

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administration of (−)-menthol on nasal resistent to air
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Eichberger, G., Penn, G., Faber, K., Griengl, H., 1986. Large
scale preparation of (+)- and (−)-endo-norbornenol by
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Harri, E., Loeffler, W., Sigg, H.P., Stahelin, H., Tamm, C.H.,
in organic media, as in aqueous media in which
there is bulk water and water tightly bound to the
enzyme, there is bulk solvent and bound solvent.
However this model cannot be of predictive value
because of the great number of possible solvent-
enzyme complexes (equal to the number of the
solvent used) and because each complex might
behave different depending on the nature of the
substrate used.
8
1971. Uber die isoliekung neuer stoffwechselprodukte aus
penicillium brefeldianum dodge. Helv. Chim. Acta 46,
1235–1243.
Honda, M., Hirata, K., Sucoka, H., Katsuki, T., Yamaguki,
M., 1981. A synthesis of (9)-Brefeldin A. Tetrahedron
Lett. 22, 2679–2682.
Kaverna, Sundholm, O., 1993. Lipase catalysis in resolution of
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Acknowledgements
This work was supported by EC Framework
Program IV-Biotechnology (1994–1998)
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