Activity and enantioselectivity of the hydroxynitrile lyase MeHNL in dry organic solvents

Article abstract of DOI:10.1002/chem.201000487

Water concentration affects both the enantioselectivity and activity of enzymes in dry organic media. Its influence has been investigated using the hydrocyanation of benzaldehyde catalyzed by hydroxynitrile lyase cross-linked enzyme aggregate (A/eHNL-CLEA) as a model reaction. The enzyme displayed higher enantioselectivity at higher water concentration, thus suggesting a positive effect of enzyme flexibility on selectivity. The activity increased on reducing the solvent water content, but drastic dehydration of the enzyme resulted in a reversible loss of activity.

Full text of DOI:10.1002/chem.201000487

DOI: 10.1002/chem.201000487
Activity and Enantioselectivity of the Hydroxynitrile Lyase MeHNL in Dry
Organic Solvents
[
a]
[c]
[b]
Monica Paravidino, Menno J. Sorgedrager, Romano V. A. Orru,* and
[
a]
Ulf Hanefeld*
Abstract: Water concentration affects both the enantioselectivity and activity of
enzymes in dry organic media. Its influence has been investigated using the hydro-
cyanation of benzaldehyde catalyzed by hydroxynitrile lyase cross-linked enzyme
aggregate (MeHNL-CLEA) as a model reaction. The enzyme displayed higher
enantioselectivity at higher water concentration, thus suggesting a positive effect
of enzyme flexibility on selectivity. The activity increased on reducing the solvent
water content, but drastic dehydration of the enzyme resulted in a reversible loss
of activity.
Keywords: enzyme catalysis · hy-
droxynitrile lyase · immobilization ·
organic solvents · reaction medium ·
water
Introduction
as a sufficient guarantee of “dry” conditions. Several exam-
[8–16]
ples, however,
have indicated that residual water can
The use of nonaqueous media constitutes a major break-
through in effective biocatalysis, since it enables the applica-
tion of a tremendous range of substrates that do not dissolve
still influence both the activity and enantioselectivity of en-
zymes; furthermore, this effect is rather difficult to predict.
Thus, a better understanding of the influence of water on
enzymes in organic media is highly desirable. Studying the
hydroxynitrile lyase-catalyzed synthesis of cyanohydrins is
particularly suitable to gain this understanding, since water
is not a reactant in this versatile enantioselective reaction.
Moreover, the use of organic solvents as reaction media for
the hydrocyanation has been reported as an efficient strat-
egy for suppressing uncatalyzed racemic cyanohydrin forma-
tion. As a result, biphasic systems consisting of an aqueous
[1]
in water. Moreover, organic solvents can significantly in-
[
1]
crease the potential activity and selectivity of enzymes,
and even invert their enantioselectivity,
[1,2]
thus enabling
new applications in synthesis. For these reasons, the use of
[3–7]
enzymes in organic media, although almost 100 years old,
is still an active research field. However, while many suc-
cessful applications have been described, a certain oversim-
plification of such systems is apparent from the relevant lit-
erature. For example, the role of residual water is often ne-
glected and the “organic” nature of the solvent is regarded
[17–22]
buffer and an organic solvent,
and so-called microaqu-
[23]
eous systems, are commonly used.
[16]
[9,11–16]
Hydrolases,
and especially lipases,
have often
been investigated in studies of the effect of residual water
on enzymes in organic solvents, probably due to their popu-
larity in organic synthesis. However, water is the natural
substrate for these enzymes, and this complicates the study
of hydrolase-catalyzed reactions; that is to say, they are not
ideal systems for investigating the influence of water on the
activity and selectivity of enzymes. Furthermore, it is known
that residual water can promote undesired side reactions
when hydrolases are involved. For example, it can cause par-
tial hydrolysis of both acyl donor and products in the lipase-
[
a] Dr. M. Paravidino, Dr. U. Hanefeld
Gebouw voor Scheikunde, Afdeling Biotechnologie
Technische Universiteit Delft
Julianalaan 136, 2628 BL Delft (The Netherlands)
Fax : (+31)15 278 1415
E-mail: U.Hanefeld@tudelft.nl
[
b] Prof. Dr. R. V. A. Orru
Department of Chemistry and Pharmaceutical Sciences
Vrije Universiteit Amsterdam
De Boelelaan 1083, 1081 HV Amsterdam (The Netherlands)
Fax : (+31)20 598 74 88
[24]
mediated acylation of alcohols.
The resulting release of
E-mail: rva.orru@few.vu.nl
carboxylic acid reduces the yield and affects the enantiose-
lectivity. In addition, it can severely hamper dynamic kinetic
[
c] Dr. M. J. Sorgedrager
CLEA Technologies B.V.
Delftechpark 34, 2628 XH Delft (The Netherlands)
[25]
resolutions. The importance of such water-induced reac-
7596
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 7596 – 7604

FULL PAPER
tions in organic mixtures is supported by ample evi-
The influence of water on the hydrocyanation was studied
[26–29]
dence.
The hydrocyanation of carbonyl compounds catalyzed by
over a wide range of initial water activities (a ). Since the
W
potential release of gaseous HCN in the course of the reac-
[30]
[53]
hydroxynitrile lyases (HNLs, also known as oxynitrilases)
tion does not allow water activity equilibration, we refer
is a reaction of equal importance to hydrolase-catalyzed
instead to specified water concentrations, these being direct-
ly related to the thermodynamic activity. Each solvent was
esterification, since it is a CꢀC bond-forming process. No
water is involved as a reactant in this reaction; in addition,
the enantioselectively prepared cyanohydrins display great
used either as anhydrous grade (that is, as sold by Sigma–
ꢁ
Aldrich in Sure/Seal bottles and hereinafter referred to as
[31]
potential as chiral building blocks in organic synthesis.
“dry”), water-saturated, or pre-equilibrated with a salt hy-
drate pair. The choice of such conditions allowed us to oper-
ate with significantly different values of initial water concen-
trations.
Nowadays, HNLs are the tool of choice for the enantioselec-
tive synthesis of these valuable compounds, even on an in-
[
31–36]
dustrial scale.
Recent advances in immobilization tech-
[37]
nology have enabled the application of HNLs in mono-
phasic organic systems. Enzymes immobilized on different
supports or as CLEAs (Cross-Linked Enzyme Aggregates)
display enhanced stability towards organic solvents and can
easily be recycled.
tivities compared to those achieved with the free HNLs
Salt pairs are known to serve as “water activity buf-
[12,13,54,55]
fers”:
when both species of a certain pair are simul-
taneously present, the water activity of the system reaches
the equilibrium value. In this study, the following three sys-
tems were chosen:
[38–41]
In many cases, higher enantioselec-
[42–49]
have been observed under these conditions.
However,
Na HPO =Na HPO ꢁ 2 H O, a ¼ 0:16
2
4
2
4
2
W
the influence of water on these reactions in organic media is
not well understood.
ðabbreviation : 0 ꢀ 2Þ
In this study, the enzymatic hydrocyanation of benzalde-
hyde in organic media has been chosen to investigate the
effect of water on both enzyme activity and enantioselectivi-
ty. The water concentration of the reaction mixture has
been measured both at the beginning and at the end of the
reaction. The obtained results highlight the importance of
correlating water concentration with both enzyme activity
and selectivity.
Na HPO ꢁ 2 H O=Na HPO ꢁ 7 H O, a ¼ 0:57
2
4
2
2
4
2
W
ðabbreviation : 2 ꢀ 7Þ
Na HPO ꢁ 7 H O=Na HPO ꢁ 12 H O, a ¼ 0:80
2
4
2
2
4
2
W
ðabbreviation : 7 ꢀ 12Þ
The use of such salt pairs allowed us to set the initial water
activity aW at three different values. In experiments with salt
pairs, MeHNL-CLEA was stirred for 1 h in the dry solvent
in the presence of a 1:1 (w/w) mixture of each pair suspend-
ed in a tea-bag approach. Preliminary experiments had
shown that this time was sufficient to reach equilibrium. In
experiments with “dry” or water-saturated solvents, no salt
pairs were added, and MeHNL-CLEA was suspended in
these solvents for 1 h. The water concentration in each case
was then measured by Karl Fischer titration and the salt
pair (when present) was removed from the system immedi-
ately thereafter. After adding the substrate (1) and the inter-
nal standard (triisopropylbenzene) to the enzyme suspen-
sion, the hydrocyanation was started by the addition of ace-
tone cyanohydrin (2). In all experiments, the reaction was
followed over 24 h by chiral HPLC. The final water concen-
tration was measured after this time. For reactions in
octane, in which mandelonitrile is not soluble, ee progression
could not be followed. Only after 24 h, the product was dis-
solved by the addition of isopropanol and the final ee was
determined.
Results and Discussion
The (S)-selective hydroxynitrile lyase from cassava,
MeHNL, is widely used in asymmetric cyanohydrin synthe-
[
31]
[50]
sis; recombinant expression guarantees that it is readily
available. To ensure optimal performance in organic sol-
vents, MeHNL-CLEA was chosen, as it is a very robust bio-
[
44,45]
catalyst,
bility in organic media containing only very low percentages
often not even defined) of water (microaqueous sys-
showing both high activity and outstanding sta-
(
[23]
tems ).
As a model reaction, the MeHNL-CLEA-mediated trans-
hydrocyanation of benzaldehyde with acetone cyanohydrin
was chosen (Scheme 1). Since the hydrophobicity of the sol-
vent may also play an important role with regard to enzyme
[51,52]
selectivity and activity,
lected: methyl tert-butyl ether (MTBE, logP=0.94), toluene
logP=2.73), and octane (logP=5.15).
three different solvents were se-
(
MTBE as solvent: MTBE stored under nitrogen in Sure/
ꢁ
Seal bottles has a very high water concentration (Table 1),
especially in comparison with toluene and octane of identi-
cal grade (Tables 3 and 4). Addition of any salt hydrate to
dry MTBE to set the aW increases the amount of water dis-
solved in the reaction medium. However, when both
MeHNL-CLEA and the salt pair are present, the amount of
water dissolved in the system is lower than that when only
Scheme 1. MeHNL-catalyzed hydrocyanation of benzaldehyde in differ-
ent organic solvents.
Chem. Eur. J. 2010, 16, 7596 – 7604
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7597

R. V. A. Orru, U. Hanefeld et al.
the salt pair is present. This indicates that the active but cat-
alytically not used enzyme alters the water concentration
that corresponds to the given aW.
On the other hand, when the water concentration is in-
creased above the optimum level, the activity is reduced
once more. This might be due to a high concentration of
water in the active site that
needs to be displaced by cya-
Table 1. Initial and final water concentrations for reactions in MTBE.
nide for the reaction to take
“Dry”
Sat.
0–2
2–7
7–12
[66]
place.
In all cases, the reac-
[
a]
[b]
[d]
water concentration of solvent [ppm]
water concentration of solvent + salt pair [ppm]
initial water concentration of the reaction mixture [ppm]
2400
–
7700
13100
–
13000
16300
–
–
–
tion was found to be faster
when the salt pairs were em-
[
c]
[c]
[e]
[c]
[e]
4000
2000
8200
5000
3200
9600
8200
3600
[
d]
[e]
[f]
final water concentration of the reaction mixture [ppm]
10300
12300 ployed to adjust the aW before
the reaction. Systems with
[
a] Measured for a sample of anhydrous solvent from Sigma–Aldrich. [b] Measured for a sample of water-satu-
rated solvent. [c] In a separate experiment, the salt pairs were suspended in the anhydrous solvent and the higher initial water concentra-
system was stirred for 1 h before measuring the water concentration. [d] Measured after stirring the CLEA in tions
(water-saturated
and
the solvent for 1 h. [e] Measured after stirring the CLEA and the salt pair in the solvent for 1 h. [f] Measured
“
dry” MTBE) displayed re-
for the reaction mixture after 24 h.
markably lower initial rates.
After 24 h, all of the systems
A beneficial influence of high initial water concentration
on enzyme enantioselectivity was clearly observed
had reached the conversion equilibrium value, and no fur-
ther reaction was observed.
(
Figure 1). The highest ee values for (S)-mandelonitrile were
obtained in water-saturated MTBE (initial water concentra-
tion about 13000 ppm), while the use of either “dry” solvent
or salt pairs in order to lower the initial water activity result-
ed in a significant decrease in the enantioselectivity. These
results are in line with the trends observed for HbHNL on
[53]
[56]
Celite
and for subtilisin;
interestingly, the opposite
trend was observed by Klibanov for subtilisin Carlsberg and
[57]
a-chymotrypsin.
The enhancement of enantioselectivity
with increasing water concentration observed in our study
can be attributed to the increase in internal enzyme flexibili-
ty; indeed, the latter is known to increase with water con-
[1,58–65]
centration.
flexibility and selectivity is not always straightforward for
However, the correlation between enzyme
[53]
different enzymes. Moreover, when studying the effect of
enzyme flexibility on selectivity at different water concen-
trations, any other possible effect (i.e., a solvent effect)
should be ruled out. Therefore, experiments comparing the
enantioselectivity of an enzyme at various water concentra-
Figure 1. Variation in ee of (S)-mandelonitrile (3) in the MeHNL-CLEA-
catalyzed hydrocyanation in MTBE with different water concentrations
(
&
dry; * sat.; * 0–2; & 2–7; ~ 7–12).
[56,57]
tions should always be referred to the same medium,
was done here.
as
When the reaction was allowed to proceed over an ex-
tended period of time (24 h), a decrease in ee was observed
in all cases. A reference experiment in which the substrate
was allowed to stir without MeHNL-CLEA in the presence
of either acetone cyanohydrin or HCN solution in an organ-
ic solvent showed that no mandelonitrile was formed, which
rules out the occurrence of a racemic background reaction.
On the other hand, MeHNL, like all HNLs, not only catalyz-
es the formation of one enantiomer, but also its degradation
[30,44,47]
to the prochiral carbonyl compound.
Therefore, a de-
crease in ee over time can be ascribed to the ability of the
enzyme to speed up the racemization process in this
manner. This effect has also been observed in toluene (Fig-
ures 1 and 5).
The influence of water concentration on the conversion in
MTBE is significant (Figures 2 and 3): at low water concen-
tration, insufficient hydration of the enzyme lowers the ac-
Figure 2. Conversion of 1 in the MeHNL-CLEA-catalyzed synthesis of
(
S)-mandelonitrile (3) in MTBE with different water concentrations (&
[8]
tivity. This is in line with results described by Costes et al.
dry; * sat.; * 0–2; & 2–7; ~ 7–12).
7598
www.chemeurj.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 7596 – 7604

Enzyme Action in Organic Media
FULL PAPER
Figure 4. Conversion of
1
and ee of (S)-mandelonitrile (3) in the
Figure 3. Effect of water concentration on the conversion of 1 in the
MeHNL-CLEA-catalyzed synthesis of (S)-mandelonitrile (3) in MTBE
MeHNL-CLEA-catalyzed hydrocyanation in MTBE with and without
the salt pair Na HPO ·2H O/Na HPO ·7H O in the reaction medium
with salt: ~ =conv., * =ee; without salt: * =conv.,
=ee).
(
* 1 h; & 2 h; ~ 3 h; * 4 h;
&
24 h)
2
4
2
2
4
2
(
&
The final water concentration in the MTBE highlights the
Toluene as solvent: The toluene-based systems (Table 3)
contained approximately one or even two orders of magni-
tude less water than those based on MTBE (Table 1), even
though the aW was the same when using salt pairs. In the
course of the reaction, the water concentration in each
system increased noticeably due to the continuous release of
water from MeHNL-CLEA into the solvent. Hydrocyana-
tions performed in a less polar solvent such as toluene gave
better ee values than those performed in MTBE, and
showed a less pronounced influence of the water concentra-
tion on the enantioselectivity (Figure 5). As in MTBE, how-
ever, the highest enantioselectivities were observed in the
systems with the highest water concentrations, that is, “dry”
and water-saturated toluene (Table 3). The three systems
containing a salt pair showed very similar behaviour and af-
forded slightly lower ee values.
ability of this solvent to take up water trapped in MeHNL-
CLEA in the course of the reaction (Table 1). To clarify
this, the reaction was also performed in parallel both under
the usual conditions (salts removed just before adding all of
the reagents) and by keeping the pair (Na HPO ·2H O/
2
4
2
Na HPO ·7H O) in the reaction flask using a tea-bag ap-
2
4
2
proach. Remarkably, the water concentration increased
even more, by about one order of magnitude over 24 h,
when the salt pair was allowed to remain in the reaction
mixture (Table 2). This unexpected effect resulted only in a
minor change of rate, and did not affect the ee (Figure 4).
However, when MeHNL-CLEA was suspended in MTBE
and stirred for 24 h in the presence of the same salt pair, but
without reagents (Table 2, control experiment no. 1), the
water concentration was found to remain almost constant.
Thus, the increase in water concentration is a feature purely
of the reagents (Table 2, control experiment no. 2). The
latter significantly increase the polarity of the solvent; as a
consequence, the release of water by the enzyme and the
salt pair is enhanced, and the water concentration corre-
sponding to the given aW value (which remains constant) is
increased accordingly.
Considering the conversions, they were slightly lower than
those in MTBE, even though an inverse correlation between
enzymatic activity and solvent hydrophilicity has been de-
[62,67]
scribed for other enzymes.
As in MTBE, a positive
effect of low water concentrations on the reaction rate was
observed (Figures 6 and 7).
Octane as solvent: Choosing the hydrocarbon octane
(
Table 4) allowed us to explore
the effect of a very hydrophobic
with logP=5.15.
hydrophobicity is
Table 2. Initial and final water concentrations for (S)-mandelonitrile (3) synthesis in MTBE in the presence solvent,
and absence of Na HPO ·2H O/Na HPO ·7H O.
2
4
2
2
4
2
Medium
Salt kept during
the reaction
Salt removed
(standard conditions)
Control
experiments
1
known to affect enzyme activity
in hydrocyanations mediated by
various supported HNLs;
indeed, it was shown that in-
creasing logP in HbHNL-medi-
ated hydrocyanation resulted in
a dramatic increase in enzyme
activity. More generally, hydro-
2
[8]
[
a]
[a]
[c]
[d]
initial water concentration [ppm]
final water concentration [ppm]
3500
3200
9600
4200
3100
10100
[
b]
[b]
[c]
[d]
32000
5500
[
a] Measured after stirring the CLEA in the solvent for 1 h in the presence of the salt pair “2–7”. [b] Measured
for the reaction mixture after 24 h. [c] The CLEA was stirred in the solvent in the presence of the salt pair “2-
” but without reagents for 24 h. The water concentration was measured after 1 h and after 24 h. [d] The re-
agents were stirred in the solvent in the presence of the salt pair “2-7” but without the CLEA. The water con-
centration was measured after 1 h and after 24 h.
7
phobic
solvents
enhance
Chem. Eur. J. 2010, 16, 7596 – 7604
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7599

R. V. A. Orru, U. Hanefeld et al.
Figure 5. Variation in ee of (S)-mandelonitrile (3) in the MeHNL-CLEA-
Figure 7. Effect of water concentration on the conversion of 1 in the
MeHNL-CLEA-catalyzed synthesis of (S)-mandelonitrile (3) in toluene.
catalyzed hydrocyanation in toluene at different water concentrations (
&
dry; * sat.; * 0–2; & 2–7; ~ 7–12).
(
* 1 h; * 2 h; ~ 3 h; & 24 h)
Figure 6. Conversion of 1 in the MeHNL-CLEA-catalyzed synthesis of
S)-mandelonitrile (3) in toluene with different water concentrations (
dry; * sat.; * 0–2; & 2–7; ~ 7–12).
Figure 8. Conversion of 1 in the MeHNL-CLEA-catalyzed synthesis of
(
&
(S)-mandelonitrile (3) in octane at different water concentrations (
* sat.; * 0–2; & 2–7; ~ 7–12).
&
dry;
enzyme activity with respect to their hydrophilic counter-
parts because they are less effective in stripping the essential
enzyme-bound water. Similar effects were expected to be
prove the reaction rate significantly; rather, it did the con-
trary. For octane, however, the effect was dramatic
(Figure 8): almost full conversion was reached after 1 h. In
addition to the effect of the high logP on the enzyme, this
can be explained in terms of the poor solubility of mandelo-
nitrile and acetone cyanohydrin
[67]
manifested in our study. However, replacing MTBE (logP=
0.94) with the less polar toluene (logP=2.73) did not im-
in octane, while benzaldehyde
dissolves completely. Thus, the
Table 3. Initial and final water concentrations for reactions in toluene.
“
Dry”
Sat.
0–2
2–7
7–12
–
formed mandelonitrile tends to
separate from the reaction mix-
ture. This in situ product re-
moval shifts the equilibrium
dramatically (Figure 8). On the
other hand, the partitioning of
acetone cyanohydrin between
the aqueous and organic phases
depends on logP; the concen-
[
a]
[b]
water concentration of solvent [ppm]
water concentration of solvent + salt pair [ppm]
initial water concentration of the reaction mixture [ppm]
250
–
460
470
–
–
–
[
c]
[c]
[c]
300
370
400
180
[d]
[d]
[e]
[e]
[e]
580
40
10000
70
4400
[f]
final water concentration of the reaction mixture [ppm]
8300
10600
5600
[
a] Measured for a sample of anhydrous solvent from Sigma–Aldrich. [b] Measured for a sample of water-satu-
rated solvent. [c] In a separate experiment, the salt pairs were suspended in the anhydrous solvent and the
system was stirred for 1 h before measuring the water concentration. [d] Measured after stirring the CLEA in
the solvent for 1 h. [e] Measured after stirring the CLEA and the salt pair in the solvent for 1 h. [f] Measured
for the reaction mixture after 24 h.
7600
www.chemeurj.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 7596 – 7604

Enzyme Action in Organic Media
FULL PAPER
tration of this reagent in the water layer around the enzyme
becomes much higher in octane than in toluene or MTBE,
thus inducing higher enzymatic activity.
the binding of transition-state analogues strengthens the in-
teractions between subunits and between protein groups
and catalytic site ligands. Thus, when benzaldehyde binds to
the active site of MeHNL, and
[8]
hydrocyanation takes place, a
Table 4. Water concentrations and final (S)-mandelonitrile ee for reactions in octane.
significant
conformational
“Dry”
Sat.
0–2
–
2–7
–
7–12
–
change occurs. The enzyme
adopts a compact and rigid con-
formation, thereby reducing its
flexibility. As a consequence,
internal water molecules are re-
[
a]
[b]
water concentration of solvent [ppm]
water concentration of solvent + salt pair [ppm]
initial water concentration of the reaction mixture [ppm]
final water concentration of the reaction mixture [ppm]
final ee [%] of (S)-3
40
–
70
–
70
80
88
[
c]
[c]
[c]
50
10
50
86
50
20
40
87
60
50
[d]
[d]
[e]
[e]
[e]
50
50
86
[f]
60
89
[
a] Measured for a sample of anhydrous solvent from Sigma–Aldrich. [b] Measured for a sample of water-satu- leased into the solvent, account-
rated solvent. [c] In a separate experiment, the salt pairs were suspended in the anhydrous solvent and the ing for the final water concen-
system was stirred for 1 h before measuring the water concentration. [d] Measured after stirring the CLEA in
the solvent for 1 h. [e] Measured after stirring the CLEA and the salt pair in the solvent for 1 h. [f] Measured
for the reaction mixture after 24 h.
[73]
tration.
These results shed
new light on the drastic loss of
activity observed for immobi-
lized HbHNL after incubation
Final ee values for mandelonitrile were determined upon
addition of isopropanol to the reaction mixture after 24 h.
These were similar to those observed in MTBE and lower
than those in toluene (Table 4; Figures 1 and 5). It is known
that enzyme enantioselectivity in nonaqueous media can
in organic solvent saturated with buffer in the presence of
[74]
phenylpropanal.
Binding of this substrate resulted in a
tight complex, which rapidly released the internal water. As
a consequence, the activity dropped. In the course of such a
reaction, the reagents, intermediates (HCN), and products
cause the solvent polarity to change. With an increase in po-
larity, the water trapped in the CLEA will escape into the
reaction mixture. The relationship between the residual
water trapped in commercial MeHNL-CLEA and its catalyt-
ic activity was highlighted by an additional experiment. By
dehydration using P O , the internal water in this CLEA
[68–71]
depend markedly on the solvent,
and in some cases
[2]
even complete inversion of enantioselectivity following a
change of solvent has been reported. MeHNL-CLEA dis-
played (S)-selectivity in all of the media screened, and only
a small decrease in the ee of (S)-mandelonitrile was ob-
served when using octane or MTBE. Such differences com-
pared to toluene can be attributed to the influence of the
solvent on the enzyme conformation.
2
5
was reduced to 4% (as determined by TGA; Figure 9). The
dehydrated enzyme showed a dramatic drop in both activity
and enantioselectivity in the hydrocyanation of benzalde-
hyde in either dry MTBE or toluene according to the de-
scribed protocol. However, the catalytic activity was reversi-
bly restored upon rehydration (decomposition of mandeloni-
An observation made for all of the solvents was a signifi-
cant increase in the water concentration in the reaction mix-
ture after 24 h. This was particularly prominent for MTBE
and toluene. The water concentrations of all reaction com-
ponents were then measured (Table 5), and were found to
be considerable. The water trapped in commercially avail-
able MeHNL-CLEA was determined by thermogravimetric
analysis (TGA) to be 38% by weight (Figure 9). The extent
to which this water is released into the reaction medium
varies greatly depending on the solvent; toluene and espe-
cially MTBE showed a marked ability to take up the water
from the immobilized enzyme. The release of water from
the enzyme most probably depends on the catalytic activity
of the enzyme and the polarity changes of the solvent due
[75]
trile in aqueous buffer). This result is analogous to the re-
[76]
activation upon rehydration described by Sym for porcine
pancreas lipase. It is thus clear that the internal water is es-
sential for the activity of MeHNL-CLEA, as has previously
[77]
been described for other enzymes. The deleterious effect
of dehydration on the catalytic activity of enzymes is a
[67,78,79]
common feature of different proteins,
consequence of significant denaturation.
and is often a
The water concentrations of several free or immobilized
commercial enzymes were also measured by TGA and com-
pared with that of MeHNL-CLEA (Figure 9). The latter
contains a remarkably higher amount of water than Candida
rugosa lipase (both free and immobilized as CLEA) or com-
mercial Novozym 435, but the amount is comparable to that
held by the CLEA of the (R)-selective and structurally un-
[72,73]
to the presence of the reagents. It has been shown
that
Table 5. Water concentrations of each of the reaction components.
System
Water concentration [ppm]
[31]
[
a]
MTBE
MeHNL-CLEA
200
7800
400
5100
13500
related HNL from Prunus amygdalus.
[
b]
[
c]
benzaldehyde (1)
acetone cyanohydrin (2)
[
c]
Conclusion
total
[
a] Directly from a Sure/Seal bottle. [b] The CLEA was suspended in dry
The MeHNL-CLEA-catalyzed hydrocyanation of benzalde-
hyde has proved to be an excellent model reaction for inves-
tigating the role of residual water in enzymatic transforma-
MTBE and stirred for 24 h. A sample of solvent was then injected into
the Karl Fischer titrator. [c] Samples of freshly distilled reagents were in-
jected directly into the Karl-Fischer titrator.
Chem. Eur. J. 2010, 16, 7596 – 7604
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7601

R. V. A. Orru, U. Hanefeld et al.
Enzymes: MeHNL-CLEA, PaHNL-CLEA, and Candida rugosa lipase
(
type VII)-CLEA were supplied by CLEA Technologies B.V. Candida
rugosa lipase (type VII) was purchased from Sigma Aldrich. Novo-
zym 435 was donated by Novozymes.
Enzyme activity measurement: The enzymatic activity of MeHNL-CLEA
[
75]
was measured according to reported literature procedures
and was
found to be 0.44 Umg . Samples were prepared by suspending CLEA
32 mg) in pH 6.5 phosphate buffer (3 mL).
ꢀ1
(
Chemicals: (ꢂ)-Mandelonitrile (Acros Organics, technical grade) was pu-
rified through column chromatography (PE/EtOAc 9:1 ! 3:7) prior to
use. Acetone cyanohydrin was distilled in the presence of 2% phosphoric
acid prior to use and was stored under nitrogen at 48C. Benzaldehyde, of
analytical grade, was always distilled prior to use and was stored under
nitrogen at 48C. Anhydrous MTBE (99.8%), toluene (99.8%), and
octane (>99%) were purchased from Sigma–Aldrich.
Analytical methods: The course of each enzyme reaction was followed
by chiral HPLC analysis at 408C using a Waters system (Waters 486 UV
detector, Waters 515 pump, and Waters 717+ injector) equipped with a
Chiralcel OB-H column from Daicel (4.5 mm ꢂ 250 mm) and using n-
heptane/2-propanol (95:5) as solvent (flow rate: 1 mLmin ). Retention
times: 3.68 min (triisopropylbenzene), 7.00 min (benzaldehyde),
Figure 9. TGA of different enzyme preparations in nitrogen at a heating
ꢀ
1
rate of 108Cmin
.
ꢀ
1
1
5.81 min ((R)-mandelonitrile), 16.83 min ((S)-mandelonitrile). Water
tions in organic solvents. The immobilized enzyme was
stable in all of the screened organic solvents and a slight
positive effect of increasing logP on the product ee was ob-
served. Remarkably higher conversions achieved in octane
may be partly attributed to separation of the product during
the reaction, which shifts the reaction equilibrium.
concentrations in solvents and reaction mixtures were determined by
Karl Fischer titration using a Metrohm 831 KF coulometer equipped
with a generator electrode with diaphragm, according to the manual pro-
vided (determination range: 10 mg–200 mg H
reproducibility: ꢂ3 mg in the range 10–1000 mg H
2
O; resolution: 0.1 mg H
2
O;
2
O, 0.3% or better for
values above 1000 mg). All measurements were performed in duplicate
and the numbers in the tables are mean values. Water trapped in
MeHNL-CLEA was measured by thermogravimetry using a Perkin–
Elmer TGA7 thermogravimetric analyzer. The measurements were per-
formed under nitrogen atmosphere in the range 25–6258C at a heating
It has clearly been demonstrated that the concentration of
water in an organic medium should not be neglected, as it
can affect both activity and selectivity. In the case of
MeHNL-CLEA, an increase in the water concentration for
a given solvent induced higher enzyme enantioselectivity. A
direct correlation between enzyme flexibility and enantiose-
lectivity can be inferred. This conclusion is at variance with
ꢀ
1
rate of 108Cmin . The initial sample mass was always in the range 4–
2 mg.
1
Blank experiment: Benzaldehyde (100 mL, 1 mmol) was placed first
under vacuum (oil pump) and subsequently under nitrogen, and finally
dissolved in dry MTBE (1 mL). The nitrogen line was then closed in
order to prevent HCN leakage. Triisopropylbenzene (84 mmol, 20 mL)
was added and an HPLC sample to determine the initial conditions was
prepared by taking 10 mL of reaction mixture, diluting it with n-heptane/
[57]
that drawn by Rariy and Klibanov for subtilisin Carlsberg
and a-chymotrypsin.
In general, higher activity was observed at relatively low
water levels. However, thorough drying of the enzyme prior
to the reaction did lead to reversible deactivation. The
amount of this residual water in commercial MeHNL-
CLEA was found to be as high as 38% (w/w). Its role is es-
sential in ensuring enzymatic activity when performing hy-
drocyanations in dry organic solvents. The release of
enzyme-bound water into the solvent accounts for the ob-
served increase of water concentration during the reaction.
For a better understanding of enzymatic reactions in or-
ganic solvents, the water contents of enzyme preparations
and reagents should always be taken into account. Further-
more, deeper insight into the complex role of water can be
obtained by measuring the final water concentration and
comparing it with the initial one.
2
-propanol (95:5), and filtering before injection. After 20 min, the initial
water concentration was also measured. The reaction was initiated by the
addition of a 1.05m solution of HCN in diisopropyl ether (6 mL, 6 equiv)
and was monitored by chiral HPLC over one day. Samples (10 mL) were
withdrawn at regular intervals (1, 2, 3, 4, 24 h) according to the described
procedure. The final water concentration was determined after 24 h.
General procedure for the enzymatic hydrocyanation in dry and water-sa-
ꢀ
1
turated solvents: MeHNL-CLEA (0.44 Umg , 15 U) was placed first
under vacuum (oil pump) and subsequently under nitrogen, and finally
suspended in solvent (1.7 mL). The nitrogen line was then closed in order
to prevent HCN leakage. A sample (10 mL) was taken after 1 h by means
of a syringe to measure the initial water concentration. Benzaldehyde
(
(
1.0 mmol, 100 mL) and the internal standard triisopropylbenzene
84 mmol, 20 mL) were added and after a few minutes an HPLC sample
to determine the initial conditions was prepared by taking 10 mL of the
reaction mixture, diluting it with n-heptane/2-propanol (95:5), and filter-
ing before injection. The reaction was initiated by the addition of acetone
cyanohydrin (560 mL, 6 equiv) and monitored by chiral HPLC over one
day. Samples (10 mL) were withdrawn at regular intervals (1, 2, 3, 4, 24 h)
according to the described procedure. The final water concentration was
determined after 24 h.
General procedure for the enzymatic hydrocyanation in the presence of a
salt pair: The salt pairs (0.5 g of each salt) were weighed onto a filter
paper (Rotilabo, Ø 55 mm), which was folded and then added to a flask
already containing MeHNL-CLEA (0.44 Umg , 15 U). The flask was
placed under vacuum and then nitrogen was admitted. The solvent
(MTBE, toluene, or octane; each stored under nitrogen) was then added.
The nitrogen line was closed and the system was stirred at room tempera-
Experimental Section
ꢀ
1
CAUTION: All procedures involving HCN were performed in a well-
ventilated fume-hood equipped with an HCN detector. HCN-containing
wastes were neutralized using commercial bleach and stored independ-
ently over a large excess of bleach for disposal.
7602
www.chemeurj.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 7596 – 7604

Enzyme Action in Organic Media
FULL PAPER
ture for 1 h; a sample was then taken by means of a syringe to determine
the initial water concentration by Karl Fischer titration. The salts were
then removed. Benzaldehyde (1.0 mmol, 100 mL) and triisopropylbenzene
[30] L. Rosenthaler, Biochem. Z. 1908, 14, 238–253.
[31] J. Holt, U. Hanefeld, Curr. Org. Synth. 2009, 6, 15–37.
[32] F. Effenberger, S. Fçrster, H. Wajant, Curr. Opin. Biotechnol. 2000,
11, 532–539.
(
84 mmol, 20 mL) were added and, after a few minutes, a HPLC sample to
determine the initial conditions was prepared by taking 10 mL of the reac-
tion mixture, diluting it with n-heptane/2-propanol (95:5), and filtering
before injection. The reaction was initiated by the addition of acetone cy-
anohydrin (560 mL, 6 equiv) and monitored by chiral HPLC over one
day. Samples (10 mL) were withdrawn at regular intervals (1, 2, 3, 4, 24 h)
according to the described procedure. The final water concentration was
determined after 24 h.
[33] H. Griengl, H. Schwab, M. Fechter, Trends Biotechnol. 2000, 18,
252–256.
[34] D. V. Johnson, A. A. Zabelinskaja-Mackova, H. Griengl, Curr. Opin.
Chem. Biol. 2000, 4, 103–109.
[35] T. Purkarthofer, W. Skranc, C. Schuster, H. Griengl, Appl. Micro-
biol. Biotechnol. 2007, 76, 309–320.
[36] F. Effenberger, S. Fçrster, C. Kobler, in Biocatalysis in the Pharma-
ceutical and Biotechnology Industries (Ed.: R. N. Patel), CRC Press,
New York, 2007, pp. 677–698.
[
[
[
[
37] U. Hanefeld, L. Gardossi, E. Magner, Chem. Soc. Rev. 2009, 38,
53–468.
38] I. W. C. E. Arends, R. A. Sheldon, U. Hanefeld, Green Chemistry
and Catalysis, Wiley-VCH, Weinheim, 2007.
4
Acknowledgements
39] L. Veum, U. Hanefeld, A. Pierre, Tetrahedron 2004, 60, 10419–
M.P. acknowledges financial support from the IBOS program of NWO/
ACTS (project no. 053.63.304). Ben Norder (TU Delft) is gratefully ac-
knowledged for performing the TG analyses. Thanks are due to Dr. Vin-
cenzo R. Calderone for helpful discussions.
1
0425.
40] M. H. Fechter, H. Griengl, in Enzyme Catalysis in Organic Solvents,
Vol. 2 (Eds.: K. Drauz, H. Waldmann), Wiley-VCH, Weinheim,
2
002, pp. 974–989.
[
[
[
[
[
41] J. N. Andexer, J. V. Langermann, U. Kragl, M. Pohl, Trends Biotech-
nol. 2009, 27, 599–607.
42] E. Wehtje, P. Adlercreutz, B. Mattiasson, Biotechnol. Bioeng. 1990,
[
[
[
[
[
[
[
[
1] A. M. Klibanov, Nature 2001, 409, 241–246.
2] S. Tawaki, A. M. Klibanov, J. Am. Chem. Soc. 1992, 114, 1882–1884.
3] E. Bourquelot, M. Bridel, Ann. Chim. Phys. 1913, 29, 145–218.
4] E. A. Sym, Biochem. J. 1930, 24, 1265–1281.
5] E. A. Sym, Biochem. Z. 1931, 230, 19–50.
6] E. A. Sym, Biochem. Z. 1933, 258, 304–324.
3
6, 39–46.
43] F. Effenberger, B. Hçrsch, S. Fçrster, T. Ziegler, Tetrahedron Lett.
990, 31, 1249–1252.
44] F. L. Cabirol, U. Hanefeld, R. A. Sheldon, Adv. Synth. Catal. 2006,
48, 1645–1654.
1
3
7] E. A. Sym, Enzymologia 1936, 1, 156–160.
8] D. Costes, E. Wehtje, P. Adlercreutz, Enzyme Microb. Technol.
45] A. Chmura, G. M. van der Kraan, F. Kielar, L. M. van Langen, F.
van Rantwijk, R. A. Sheldon, Adv. Synth. Catal. 2006, 348, 1655–
1
999, 25, 384–391.
1
661.
[
9] H.-E. Hçgberg, H. Edlund, P. Berglund, E. Hedenstrçm, Tetrahe-
dron: Asymmetry 1993, 4, 2123–2126.
[
[
[
46] C. Mateo, J. M. Palomo, L. M. van Langen, F. van Rantwijk, R. A.
Sheldon, Biotechnol. Bioeng. 2004, 86, 273–276.
47] C. Roberge, F. Fleitz, D. Pollard, P. Devine, Tetrahedron Lett. 2007,
[
10] C. Orrenius, T. Norin, K. Hult, G. Carrea, Tetrahedron: Asymmetry
995, 6, 3023–3030.
11] J. Bodnꢃr, L. Gubicza, L. P. Szabꢄ, J. Mol. Catal. 1990, 61, 353–361.
12] P. J. Halling, Enzyme Microb. Technol. 1994, 16, 178–206.
13] L. Kvittingen, B. Sjursnes, T. Anthonsen, P. Halling, Tetrahedron
1
4
8, 1473–1477.
[
[
[
48] M. Avi, H. Griengl, in Organic Synthesis with Enzymes in Non-
Aqueous Media (Eds.: G. Carrea, S. Riva), Wiley-VCH, Weinheim,
2
008, pp. 211–226.
1
992, 48, 2793–2802.
[
[
49] L. M. van Langen, R. P. Selassa, F. van Rantwijk, R. A. Sheldon,
Org. Lett. 2005, 7, 327–329.
50] S. Fçrster, J. Roos, F. Effenberger, H. Wajant, A. Sprauer, Angew.
Chem. 1996, 108, 493–494; Angew. Chem. Int. Ed. Engl. 1996, 35,
[
[
[
[
[
[
[
14] V. Lꢅonard-Nevers, Z. Marton, S. Lamare, K. Hult, M. Graber, J.
Mol. Catal. B 2009, 59, 90–95.
15] E. Wehtje, J. Kaur, P. Adlercreutz, S. Chand, B. Mattiasson, Enzyme
Microb. Technol. 1997, 21, 502–510.
16] E. P. Hudson, R. K. Eppler, J. M. Beaudoin, J. S. Dordick, J. A.
Reimer, D. S. Clark, J. Am. Chem. Soc. 2009, 131, 4294–4300.
17] F. Effenberger, T. Ziegler, S. Fçrster, Angew. Chem. 1987, 99, 491–
92; Angew. Chem. Int. Ed. Engl. 1987, 26, 458–460.
18] T. T. Huuhtanen, L. T. Kanerva, Tetrahedron: Asymmetry 1992, 3,
223–1226.
19] V. I. Ognyanov, V. K. Datcheva, K. S. Kyler, J. Am. Chem. Soc. 1991,
13, 6992–6996.
4
37–439.
[
[
[
51] G. Carrea, G. Ottolina, S. Riva, Trends Biotechnol. 1995, 13, 63–70.
52] T. Ke, A. M. Klibanov, J. Am. Chem. Soc. 1998, 120, 4259–4263.
53] M. Persson, D. Costes, E. Wehtje, P. Adlercreutz, Enzyme Microb.
Technol. 2002, 30, 916–923.
4
[
54] P. J. Halling, Biotech. Technol. 1992, 6, 271–276.
1
[55] Z. Yang, D. A. Robb, Biotechnol. Techn. 1993, 7, 37–42.
[56] J. Broos, A. J. W. G. Visser, J. F. J. Engbersen, W. Verboom, A. van
Hoek, D. N. Reinhoudt, J. Am. Chem. Soc. 1995, 117, 12657–12663.
[57] R. V. Rariy, A. M. Klibanov, Biocatal. Biotransform. 2000, 18, 401–
407.
[58] P. A. Burke, R. G. Griffin, A. M. Klibanov, Biotechnol. Bioeng.
1993, 42, 87–94.
[59] T. Ke, A. M. Klibanov, Biotechnol. Bioeng. 1998, 57, 746–750.
[60] J. A. Rupley, G. Careri, in Advances in Protein Chemistry, Vol. 41
(Eds.: C. B. Anfinsen, F. M. Richards, J. T. Edsall, D. S. Eisenberg),
Academic Press, New York, 1991, pp. 37–172.
[61] S. Toba, D. S. Hartsough, K. M. Merz, J. Am. Chem. Soc. 1996, 118,
6490–6498.
[62] A. Zaks, A. M. Klibanov, J. Biol. Chem. 1988, 263, 3194–3201.
[63] A. Zaks, A. M. Klibanov, J. Biol. Chem. 1988, 263, 8017–8021.
[64] J. Partridge, P. R. Dennison, B. D. Moore, P. J. Halling, Biochim.
Biophys. Acta 1998, 1386, 79–89.
1
20] P. Zandbergen, J. v. d. Linden, J. Brussꢅe, A. v. d. Gen, Synth.
Commun. 1991, 21, 1387–1391.
[
[
21] T. Ziegler, B. Hçrsch, F. Effenberger, Synthesis 1990, 575–579.
22] H. Griengl, N. Klempier, P. Pçchlauer, M. Schmidt, N. Shi, A. A.
Zabelinskaja-Mackova, Tetrahedron 1998, 54, 14477–14486.
23] G. Lin, S. Han, Z. Li, Tetrahedron 1999, 55, 3531–3540.
24] P. Hara, U. Hanefeld, L. T. Kanerva, Green Chem. 2009, 11, 250–
56.
25] Y. X. Li, A. J. J. Straathof, U. Hanefeld, Tetrahedron: Asymmetry
002, 13, 739–743.
[
[
2
[
2
[
[
26] X.-G. Li, L. T. Kanerva, Adv. Synth. Catal. 2006, 348, 197–205.
27] M. Lundh, O. Nordin, E. Hedenstrçm, H.-E. Hçgberg, Tetrahedron:
Asymmetry 1995, 6, 2237–2244.
[
28] L. Veum, U. Hanefeld, Tetrahedron: Asymmetry 2004, 15, 3707–
3
709.
[
29] L. Veum, L. T. Kanerva, P. J. Halling, T. Maschmeyer, U. Hanefeld,
[65] J. L. Schmitke, C. R. Wescott, A. M. Klibanov, J. Am. Chem. Soc.
1996, 118, 3360–3365.
Adv. Synth. Catal. 2005, 347, 1015–1021.
Chem. Eur. J. 2010, 16, 7596 – 7604
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7603

R. V. A. Orru, U. Hanefeld et al.
[
66] K. Gruber, G. Gartler, B. Krammer, H. Schwab, C. Kratky, J. Biol.
Chem. 2004, 279, 20501–20510.
[74] D. Costes, G. Rotcenkovs, E. Wehtje, P. Adlercreutz, Biocatal. Bio-
transform. 2001, 19, 119–130.
[
[
67] A. M. Klibanov, Trends Biotechnol. 1997, 15, 97–101.
68] L. T. Kanerva, J. Vihanto, M. H. Halme, J. M. Loponen, E. K. Eur-
anto, Acta Chem. Scand. 1990, 44, 1032–1035.
[75] U. Hanefeld, A. J. J. Straathof, J. J. Heijnen, Biochim. Biophys. Acta
1999, 1432, 185–193.
[76] E. A. Sym, Biochem. J. 1936, 30, 609–617.
[77] R. K. Eppler, E. P. Hudson, S. D. Chase, J. S. Dordick, J. A. Reimer,
D. S. Clark, Proc. Natl. Acad. Sci. USA 2008, 105, 15672–15677.
[78] K. Griebenow, A. M. Klibanov, Proc. Natl. Acad. Sci. USA 1995, 92,
10969–10976.
[
[
[
[
[
69] H. Kitaguchi, P. A. Fitzpatrick, J. E. Huber, A. M. Klibanov, J. Am.
Chem. Soc. 1989, 111, 3094–3095.
70] T. Sakurai, A. L. Margolin, A. J. Russell, A. M. Klibanov, J. Am.
Chem. Soc. 1988, 110, 7236–7237.
71] C. R. Wescott, H. Noritomi, A. M. Klibanov, J. Am. Chem. Soc.
[79] K. Griebenow, A. M. Klibanov, Biotechnol. Bioeng. 1997, 53, 351–
362.
1
996, 118, 10365–10370.
72] F. Wang, W. Shi, E. Nieves, R. H. Angeletti, V. L. Schramm, C.
Grubmeyer, Biochemistry 2001, 40, 8043–8054.
73] D. H. Williams, E. Stephens, M. Zhou, Chem. Commun. 2003, 1973–
Received: February 24, 2010
Published online: May 18, 2010
1
976.
7604
www.chemeurj.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 7596 – 7604