Pressure effects on activity and selectivity of Candida rugosa lipase in organic solvents

Article abstract of DOI:10.1016/j.molcatb.2013.12.002

Even though a lot of high pressure studies on enzyme structure, stability and activity are published in the last years, just a few works deal with the influence of pressure on the enantioselectivity of an enzymatic reaction. Furthermore, a change of the reaction medium from buffer to organic solvents for high pressure studies offers some interesting advantages, like pH independency and higher sensitivity towards hydration changes. From this point of view, in the present paper the influence of high pressure on the activity and selectivity of a Candida rugosa Lipase catalyzed reaction in organic solvents was examined. The transesterification of 1-phenylpropan-2-ol with vinyl acetate was chosen as a model reaction. The reactions carried out at 50 MPa showed an increased specific activity of the lipase, independent of solvent composition, reaction temperature and water content of the solvent. An activity maximum, without deactivation, was observed in hexane at 45 C and 200 MPa. Between 50 MPa and 200 MPa a linear increase in the enantiomeric excess (ee_R) could be detected, also independent of the solvent composition, reaction temperature and water content of the reaction medium. Furthermore, if additional water was added to the reaction solvent no change of the ee_R at high pressures could be observed. This leads to the conclusion that the ee_R under pressure is probably mainly influenced by the compression state of the enzyme or by structural changes of the active center rather than by the water content of the enzyme, as it is the case at ambient pressure.

Full text of DOI:10.1016/j.molcatb.2013.12.002

Journal of Molecular Catalysis B: Enzymatic 100 (2014) 104–110
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Pressure effects on activity and selectivity of Candida rugosa lipase in
organic solvents
Daniela Herbsta,∗, Stephanie Peperb, José Francisco Fernándeza,
Wolfgang Ruckc, Bernd Niemeyera
a Helmut-Schmidt-University/University of Federal Armed Forces Hamburg, Holstenhofweg 85, Hamburg D-22043, Germany
b Bayer Technology Services GmbH, Department of Property Data and Thermodynamics, Building B 310, Leverkusen D-51368, Germany
c Leuphana University Lüneburg, Scharnhorststraße 1, Lüneburg D-21335, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 May 2013
Received in revised form 5 December 2013
Accepted 7 December 2013
Available online 15 December 2013
Even though a lot of high pressure studies on enzyme structure, stability and activity are published
in the last years, just a few works deal with the influence of pressure on the enantioselectivity of an
enzymatic reaction. Furthermore, a change of the reaction medium from buffer to organic solvents for
high pressure studies offers some interesting advantages, like pH independency and higher sensitivity
towards hydration changes. From this point of view, in the present paper the influence of high pressure on
the activity and selectivity of a Candida rugosa Lipase catalyzed reaction in organic solvents was examined.
The transesterification of 1-phenylpropan-2-ol with vinyl acetate was chosen as a model reaction. The
reactions carried out at 50 MPa showed an increased specific activity of the lipase, independent of solvent
composition, reaction temperature and water content of the solvent. An activity maximum, without
deactivation, was observed in hexane at 45 ◦C and 200 MPa. Between 50 MPa and 200 MPa a linear increase
in the enantiomeric excess (eeR) could be detected, also independent of the solvent composition, reaction
temperature and water content of the reaction medium. Furthermore, if additional water was added to
the reaction solvent no change of the eeR at high pressures could be observed. This leads to the conclusion
that the eeR under pressure is probably mainly influenced by the compression state of the enzyme or by
structural changes of the active center rather than by the water content of the enzyme, as it is the case
at ambient pressure.
Keywords:
Candida rugosa lipase
High pressure
Enzyme catalysis
Organic solvent
Selectivity
© 2013 Published by Elsevier B.V.
1. Introduction
dependencies of chemical bonds and interactions, because these
binding types differ in their sensitivity to pressure [16,17]. In
High pressure as a tool in biotechnology is becoming more and
more important in the last few years. Common applications are
found in food industry for inactivation of microorganisms [e.g., 1–3]
or spores [e.g., 4,5] and in preservation due to pressure-induced
proteolysis [6,7]. Additional applications include the production of
fine chemicals and pharmaceuticals [8]. Since high pressure not
inevitably leads to protein denaturation but rather causes only local
changes in protein structure, it can be used to analyze changes in
structure and function during the catalytic reaction. These changes
could affect the activity, selectivity and stability of the enzyme,
which was intensively discussed and reviewed over the past years
[e.g., 9–15].
general, negative reaction volumes are preferred under high
pressure (Le Chatelier principle). That means that covalent bonds
are built under high pressure because their formation leads to a
volume decrease. Furthermore, they are stabilized by the pressure.
Hydrogen bonds are likewise stabilized because their formation is
associated with a small volume reduction. Besides, the interatomic
distances are reduced which leads to shorter, more stabilized
hydrogen bonds [18]. In contrast, electrostatic interactions are
destabilized. This is due to the electrostriction which describes the
hydration of polar and charged groups as well as their dissociation
followed by hydration. Both of these effects result in a volume
decrease [19]. This effect causes higher hydration states of the
enzyme which leads to higher local flexibilities [15]. Furthermore,
hydrophobic interactions are destabilized because their formation
is associated with a positive reaction volume. In contrast, the
stacking of aromatic rings results in a volume decrease which
means that they are stabilized under pressure.
In order to describe the influence of high pressure on the
enzyme structure or more specific on the chemical interactions
within the enzyme, it is important to look at the different pressure
Because the enzyme structure is characterized by all of these
different kinds of bonds it can be concluded that the primary
∗
Corresponding author. Tel.: +49 40 6541 2747; fax: +49 40 6541 2008.
E-mail address: dherbst@hsu-hh.de (D. Herbst).
1381-1177/$ – see front matter © 2013 Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.molcatb.2013.12.002

D. Herbst et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 104–110
105
structure is pressure resistant, because it is linked by cova-
lent bonds. The secondary structure is stabilized by hydrogen
bonds, likewise resulting in a pressure resistance. At very high
pressures between 300 and 700 MPa denaturation effects of the
secondary structure can be observed [20]. The tertiary structure is
predominantly stabilized by salt bridges and disulfide bonds and
denaturation effects upon them can be detected at around 200 MPa.
For smaller proteins denaturation arises just at 400 MPa [21]. Last,
quaternary structure is stabilized by intermolecular electrostatic
and hydrophobic interactions, which are destabilized at pressures
from 50 to 200 MPa. Furthermore, oligomers dissociate into their
subunits [22].
temperature was varied from 20 to 55 ◦C to analyze the influence of
the reaction temperature. At least water was added to the reaction
mixture, to analyze the influence of the water content.
2. Materials and methods
2.1. Chemicals and enzymes
Racemic 1-phenylpropan-2-ol (PP), racemic 1-phenylethanol
(PE), vinyl acetate (VA), anhydrous hexane (Hex) and anhy-
drous tetrahydrofurane (THF) were purchased from Sigma–Aldrich
Munich, Germany. Hexane and THF were stored in septum bottles
to avoid contact with the surrounding atmosphere. The water con-
tent, determined by coulometric Karl-Fischer titration, was 0.003%
for hexane and 0.017% for tetrahydrofurane. A reliable determina-
tion of the water content of vinyl acetate was not possible because
of the molecular double bond which causes a side reaction dur-
ing the coulometric Karl-Fischer titration. Therefore vinyl acetate
was stored and handled under argon atmosphere. The racemic
product 1-phenylpropan-2-yl acetate (PPA) was prepared by chem-
ical acetylation (pyridine, acetic anhydride, 5 mg scale) from the
racemic alcohol 1-phenylpropan-2-ol as standard for gas chro-
matography (GC) analyses. All further chemicals and solvents were
of analytical grade or higher and purchased from Sigma–Aldrich
Munich, Germany or Fluka Munich, Germany. Lipase from Candida
rugosa (activity in aqueous solution: 30,000 U/g; protein content:
4%) was donated from Amano Enzyme Inc. Nagoya, Japan.
Besides the pressure influence on the enzyme structure, the
chemical reaction exhibits a pressure dependency as well. The
dependence of the equilibrium constant (K) is displayed in Eq. (1):
ꢀ
ꢁ
∂ ln K
∂p
ꢀV
RT
= −
(1)
T
with the pressure p, the temperature T, the universal gas constant
R and the difference of the reaction volumes of products and educts
ꢁV. If the difference in the reaction volume is negative, the product
formation is favored with increasing pressure. On the other hand,
if the reaction volume is positive, the formation of the educt is
preferred.
Besides the state of equilibrium, high pressure also influences
the reaction rate (k) to accommodate the reaction equilibrium (Eq.
(2)):
ꢀ
ꢁ
=/
ꢀV
2.2. Experimental procedures
∂ ln k
∂p
= −
(2)
RT
T
The substrate solution consists of the solvents hex and THF as
well as the substrates VA and PP. None of the components were
pretreated. The mixture had a total volume of 25 mL. Because VA
as the acyl donor has to be present in excess it was treated like a
solvent.
To analyze the influence of different solvent compositions
the following three different mixtures were created. Solution 1:
18.75 mL hex and 6.25 mL VA (ratio hex:THF:VA 3:0:1); solution
2: 12.5 mL hex, 6.25 mL THF and 6.25 mL VA (ratio 2:1:1); solution
3: 18.75 mL THF and 6.25 mL VA (ratio 0:3:1). All three solutions
were staggered with 0.41 mol/L of the acyl acceptor PP and stirred
for 5 min at 300 rpm to dissolve the viscous PP. The reactions were
carried out at 35 ◦C.
The reactions to investigate the influence of different reaction
temperatures were exclusively examined in pure hexane (solution
1 staggered with PP) at 20 ◦C, 35 ◦C, 45 ◦C, and 55 ◦C, respectively.
In order to investigate the role of the water content the reac-
tions were exclusively examined in pure hexane as well. Therefore,
water was added to solution 1 staggered with PP (1 ␮L water per
mL solvent). The reactions were carried out at 35 ◦C.
with ꢁV =/ as the difference in the activation volume. Here the
molar volume difference between the transition state and the
ground state is taken into account. The reaction is accelerated when
having a negative reaction volume under high pressure [23].
A wide range of fundamental research has been performed in
order to explain structural changes of the enzyme during high
pressure treatment [16,17,24]. Thereby, most of the reactions were
carried out in buffer solutions. Up to now, there are just a few publi-
cations on the impact of high pressure on enzyme activity in organic
solvents [25–28]. One advantage of using organic solvents is their
independency to the pH value. Buffer solutions exhibit a pressure
addiction of the pH value which could affect the enzyme [29]. On
the other hand, enzyme reactions in organic solvents are strongly
influenced by the water content of the solvent, and by the hydra-
tion state of the enzyme, respectively [30]. Therefore, the impact of
high pressure on the binding and dissociation of water molecules
in relation with the hydration of polar charged amino acids could
be more relevant in organic solvents. Thus, a higher sensitivity of
the protein to pressure could be expected.
Previous works, carried out in buffer solutions with Pseu-
domonas putida Benzoylformate decarboxylase, could already show
that the enantiomeric excess increases with increasing pressure
[31,32]. The aim of this work was to investigate the influence of
high pressure not only on the enzyme activity, but also on the
enantiomeric excess of the reaction. As a model reaction the trans-
esterification reaction of 1-phenylpropan-2-ol with vinyl acetate
as acyl donor to (R/S)-1-phenylpropan-2-yl acetate catalyzed by
Candida rugosa lipase was chosen. For the examined biocatalysis in
organic solvents Candida rugosa lipase is an ideal candidate, because
it is commercially available, there is no need of a cofactor and the
lipase has a broad substrate specificity [33]. Different parameters
were varied to characterize the reaction at ambient and high pres-
sure. Therefore, to investigate the influence of different solvent
compositions three solvents were tested at 35 ◦C. Furthermore, the
Due to the low reaction rates in all cases the prepared solutions
were mixed cold and after that added to the crude lipase powder
(concentration CRL 6 g/L).
For the reactions at ambient pressure the lipase-substrate-
mixture was transferred into sealed glass vials and accomplished
in a drying oven to control the reaction temperature. To ensure a
good dispersion of the lipase in the solvent, the solution was stirred
over the whole reaction time with a stirrer bar on a magnetic stirrer
(300 rpm).
For high-pressure reactions the lipase-substrate-mixture was
directly transferred into a temperature controlled high pressure
vessel with a volume of 25 mL. The pressure vessel is depicted
in Fig. 1. The temperature of the vessel is regulated through a
water jacket which is connected to a temperature control device.
A magnetic stirrer is placed underneath the vessel to ensure a

106
D. Herbst et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 104–110
Fig. 1. Temperature controlled high pressure vessel for investigations under high pressure. 1: screw hand press pump, 2: filter layer, 3: pressure gauge, 4: magnetic stirrer
plate, 5: temperature control device. V 1–V 4: valves.
constant mixing with a stirrer bar during the whole experiment
(300 rpm). The different pressures were built up via a screw pres-
sure pump filled with the corresponding solvent of any reaction.
The experiments were carried out at 50, 100 and 200 MPa, respec-
tively. After the pressure built up all valves were closed to ensure
a leak-proof vessel. The high pressure vessel is self-designed. All
components were ordered by SITEC-Sieber Engineering AG Zurich,
Switzerland.
All reactions were stopped after 48 h for one-point analysis or
after 75 h for conversion-time analyses and centrifuged at 1075 × g
for 5 min. For conversion-time analyses samples (1.5 mL) were
taken at 0, 3, 6, 24, 30, 48, 54, 72 and 75 h, respectively. All sam-
ples were staggered with an internal standard of 1-phenylethanol
(0.08 mol/L) before measurement and analyzed by GC-FID (Clarus
500, Perkin Elmer Rodgau, Germany).
The enantiomeric excess eeR [%] was calculated by Eq. (4)
cR − cS
=
eeR
(4)
cR + cS
with the concentration of the product enantiomer (R)-1-
phenylpropan-2-yl acetate cR and the concentration of the product
enantiomer (S)-1-phenylpropan-2-yl acetate cS.
3. Results and discussion
The chosen transesterification reaction catalyzed by Candida
rugosa lipase is depicted in Fig. 2.
The reaction is irreversible by the utilization of vinyl acetate
as acyl donor. The by-product vinyl alcohol undergoes a keto-
enol tautomerization to yield mainly in acetaldehyde at ambient
pressure [34]. Because the equilibrium constant for the formation
of acetaldehyde from vinyl alcohol is about 6 × 10−6 [35], it was
assumed that the influence of pressure on that equilibrium is neg-
ligible. Furthermore, acetaldehyde is known to inactivate the lipase
by forming Schiff’ bases with amino residues of lysine [36]. In Fig. 3
the conversion and the specific activity are displayed as a function
of reaction time for the used solvent compositions.
These plots show that on the one hand the conversion increases
with the reaction time, but on the other hand the specific activ-
ity decreases. This is probably due to the acetaldehyde, because
especially CRL is known to be very sensitive towards this kind
of inactivation [37]. As a compromise between high conversion,
high activities, realizable reaction rates and good dispersion condi-
tions of the heterogeneous mixture (e.g. limitation of the amount
of enzyme to be utilized) a reaction time of 48 h was chosen for
all experiments. Furthermore, mass transfer limitations were not
considered, as no agglomerations were observed.
2.3. Analytical methods
For the determination of the specific activity and the enan-
tiomeric excess eeR the samples were analyzed by GC-FID
using a chiral Hydrodex-ß-PM column (Macherey&Nagel Dueren,
Germany; column length 50 m; column diameter 0.25 mm; condi-
tions: oven temperature from 80 ◦C (initial time 1 min) to 120 ◦C
for 17 min with a heating rate of 20 ◦C/min, split ratio 1:20,
He as carrier gas). Typical retention times were tr,PE = 11.4 min,
tr,PP = 13.4 min, tr,(S)-PPA = 16.5 min, tr,(R)-PPA = 17.2 min. There was no
separation to base line for the enantiomers of 1-phenylethanol and
1-phenylpropan-2-ol.
Product and substrate concentrations were calculated with the
single point internal standard method.
The specific activity A [U/g] was calculated by Eq. (3)
cp · V
A =
(3)
t · mE
3.1. Influence of high pressure in different solvent systems
with the concentration of the product cP, the reaction volume V,
the reaction time t and the mass of the enzyme mE used for the
reaction.
In order to analyze the effect of high pressure on the cho-
sen transesterification, the reactions were carried out in hexane,

D. Herbst et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 104–110
107
Fig. 2. Transesterification of (R/S)-1-phenylpropan-2-ol with vinyl acetate.
Fig. 3. Conversion (A) and specific activity (B) of the PPA formation as a function of time in different solvent systems. Hex (᭹) (gray scale), Hex/THF 2:1 (ꢀ), THF (ꢁ). Reaction
time 48 h. Reaction temperature 35 ◦C. Abscissa starts at 0.1 MPa.
tetrahydrofurane and a mixture of both solvents at ambient pres-
sure, 50 MPa, 100 MPa, and 200 MPa, respectively. A mixture of
hexane and tetrahydrofurane was chosen to create a solvent with
a different hydrophobicity compared to the original ones. Fur-
thermore, with this choice it was possible to minimize individual
solvent characteristics from completely different solvents. The
results are demonstrated in Fig. 4. The left graph (A) displays the
specific activity as a function of pressure in the three different sol-
vent compositions. The results show that the more hydrophilic
the solvent, the lower is the activity of the enzyme. The depen-
dence is due to the well-known “stripping”-effect of hydrophilic
solvents [38]. Therefore, hydrophilic solvents strip off the essential
water molecules from the enzyme surface. As a result, the enzyme
becomes more rigid and loses activity.
By increasing pressure up to 50 MPa, the activity increases in all
solvent systems (Hex 1.6 fold; Hex/THF 6.6 fold; THF 4.5 fold, with
respect to the specific activity exhibited at atmospheric pressure).
Two main effects could be responsible for the increasing activity.
One is a stabilizing effect during the transesterification. The cat-
alytic mechanism of the lipase is characterized by the formation of
two transition states during the catalysis [39]. In the first step the
oxygen of the substrate ester (VA) is polarized by two amino acids
Fig. 4. Specific activity (A) and enantiomeric excess (B) of the PPA formation as a function of pressure in different solvent systems. Hex (ꢁ), Hex/THF 2:1 (᭹), THF (ꢂ). Reaction
time 48 h. Reaction temperature 35 ◦C. Abscissa starts at 0.1 MPa.

108
D. Herbst et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 104–110
Fig. 5. Specific activity (A) and enantiomeric excess (B) of the PPA formation as a function of pressure at different reaction temperatures. Reaction time 48 h. Abscissa starts
at 0.1 MPa.
in the active center, which results in an oxyanion. This oxyanion is
stabilized by hydrogen bonds. In a second step the catalytic active
serine attacks the carbonyl carbon of the substrate. This results in
the formation of a covalent bond. Both, hydrogen bonds and cova-
lent bonds are stabilized and their formation is preferred under
high pressure. Thus, the formation of transition states results in a
volume decrease and the reaction rate is accelerated [15].
The other effect on the increasing activity at 50 MPa could be
an increased flexibility of the enzyme under high pressure. In
organic solvents, enzymes exhibit a more packed and rigid struc-
ture compared to buffer solutions [40]. Furthermore, polar amino
acid side chains are orientated toward the protein core and develop
intramolecular electrostatic interactions [41]. At the pressure range
up to 50 MPa these interactions will disrupt due to hydration of
charged groups [10]. As a consequence, the flexibility as well as the
activity of the enzyme increases [42]. Penniston [43] postulated
already in 1971, that monomeric enzymes are stimulated in their
activity due to the application of pressure, which could be validated
by the described effect.
By increasing the pressure up to 100 MPa, the activity drops
in all solvents. At this pressure a counter-acting affect could be
more important. The volume of internal cavities reduces due to
the compression of the enzyme. Again, the enzyme becomes more
packed and structural fluctuations are reduced [44,45]. A further
pressure increase up to 200 MPa does not cause further changes
in the activity, neither increase nor decrease. Thereby the activ-
ity remains still higher compared to ambient pressure (200 MPa:
Hex 1.4 fold; Hex/THF 2.4 fold; THF 1.5 fold). Probably no enzyme
denaturation due to the application of pressure takes place due to
the nearly stationary behavior of the activity.
Despite of the higher enzyme activity, the enantiomeric excess
eeR of the reaction strongly decreases with increasing pressure
up to 50 MPa in all solvent compositions (Fig. 4(B)). Due to the
increased flexibility caused by disruption of intramolecular elec-
trostatic interactions, it can be assumed that the active center is
also accessible for the steric hindered (S)-enantiomer, which causes
a drop in the selectivity [46]. Besides, Kahlow and colleagues [47]
describe a water channel reaching from the surface of the CRL near
the substrate binding site through the enzyme ending up next to the
catalytic active histidine. This channel is filled with water molecules
with increasing pressure up to 100 MPa. This leads to a slight depo-
sition of the histidine, which results in a reduced distance to the
catalytic active group of the unfavored enantiomer. Here, the dif-
ference in the distance between the catalytic active group of the
substrate and histidine in the active center is responsible for the
selectivity of the lipase. Therefore, the reduced distance leads to a
decrease in the selectivity.
Between 50 and 200 MPa, the eeR increases in all solvents (Hex
ꢁee = 14%, Hex/THF ꢁee = 29%, THF ꢁee = 7%). This could be due to
the compression of the enzyme. The higher the pressure, the more
packed is the enzyme [48]. This has a direct influence on the acces-
sibility of the active center and hence on the eeR. The steric hindered
enantiomer does not fit into the binding pocket and the selectiv-
ity increase. Furthermore, electrostriction could play a role in the
increased selectivity. Electrostatic interactions during the substrate
binding of the steric hindered enantiomer could be weakened with
increasing pressure, which leads to a reduced reaction rate of this
enantiomer [49].
3.2. Influence of high pressure in combination with high
temperature
Pressure and temperature are described as counteracting effects
relating to the influence of water to the enzyme. Whereas the ini-
tial step for denaturation at high temperatures is a loss of essential
water molecules which leads to structural changes of the enzyme,
this loss of water is hindered under high pressure due to the pre-
ferred hydration of charged and non-polar groups [14]. This results
in an increase in the denaturation temperature with increasing
pressure. For the examination of this dependency, reactions were
carried out in hexane at 20 ◦C, 35 ◦C, 45 ◦C and 55 ◦C, respectively.
Fig. 5(A) displays the specific activity as a function of pressure at
the different reaction temperatures.
At ambient pressure the lipase exhibited the highest activity at
35 ◦C, whereas the lowest activity was observed at 55 ◦C. Under
these conditions, production inhibition could be excluded as over a
reaction time of 72 h the lipase showed the highest activity at 35 ◦C
and the lowest at 55 ◦C (data not shown).
The pressure/activity profile for 20 ◦C as well as 35 ◦C is similar,
with the highest activity at 50 MPa (2.3 fold and 1.6 fold for 20 ◦C
and 35 ◦C, with respect to the specific activity exhibited at ambient
pressure). A slight decrease in activity could be observed between
50 and 200 MPa. At 45 ◦C the enzyme activity increases with pres-
sure up to 200 MPa (4.8 fold) and no decrease could be detected
(with respect to the error bars at 25 and 50 MPa). At 55 ◦C an intense
increase of activity with increasing pressure up 50 MPa could be
observed (6.9 fold), which was stable up to 100 MPa. However, at
200 MPa the activity drops sharply.
In general enzymes exhibit a bell-shaped pressure-temperature
dependency [17,24], which describes an optimum at a definite pres-
sure/temperature combination. That means that the temperature
where the enzyme is still native will rise with rising pressure up
to a certain point. Furthermore, the native state of enzyme under
pressure also changes with changing temperature.

D. Herbst et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 104–110
109
Fig. 6. Specific activity (A) and enantiomeric excess (B) of the PPA formation as a function of pressure under untreated and water-containing conditions. Untreated (ꢁ), 1 ␮L
water per mL solvent (᭹). Reactions were carried out in hexane. Reaction time 48 h. Reaction temperature 35 ◦C. Abscissa starts at 0.1 MPa.
For the examined reaction the temperature optimum at atmo-
spheric pressure was determined at 35 ◦C, afterwards the activity
decreases at higher temperatures. The temperature optimum
increased which increasing pressure up to 55 ◦C at 100 MPa and
decreased again to 45 ◦C at 200 MPa. The highest activity compared
to all conditions was observed at 45 ◦C and 200 MPa.
discussed influence of high pressure on the hydration of the
enzyme is reasonable for the higher increase under high pressure
conditions.
The higher flexibility of the enzyme due to the higher water
content causes a drop in the enantiomeric excess at atmospheric
pressure (Fig. 6 (B)). Interestingly, this effect was no longer
present under high pressure. The eeR for the untreated and water-
containing samples are nearly the same (untreated ꢁee: 14%,
water-containing ꢁee: 15% between 50 and 200 MPa). Whereas,
the water-content of the solvent and moreover of the enzyme has
a strong influence on the selectivity at ambient pressure, these
results indicate that this effect is strongly reduced at high pres-
sures. It seems that under high pressure the hydration state of the
enzyme does not influence the lipase selectivity in the same way
as it does at ambient pressure. The compression of the protein or
structural changes of the tertiary structure or rather of the active
center probably the more dominating effects.
The enantiomeric excess as a function of pressure at different
reaction temperatures is displayed in Fig. 5(B). At atmospheric pres-
sure, the highest eeR could be observed at 55 ◦C. Due to a higher
solubility of water in hexane at higher temperatures [e.g. 50,51],
essential water molecules are stripped off from the enzyme surface.
In other words, the stripping-effect of hexane grows with increas-
ing temperature. This leads to a more rigid enzyme and in turn to an
increase in selectivity, as it was shown in our previous work [52]. At
50 MPa the selectivity drops for all reaction temperatures, whereas
nearly no difference between 20 ◦C (eeR 9%), 45 ◦C and 55 ◦C (both
8%) could be observed. Interestingly, in the pressure range between
50 and 200 MPa the enantiomeric excess increases at all reaction
temperatures to approximately the same extent (20 ◦C ꢁee = 15%,
35 ◦C ꢁee = 14%, 45 ◦C ꢁee = 11%, 55 ◦C ꢁee = 7%) and only slight
differences in the eeR between the different reaction temperatures
are observed. It seems that there is no temperature dependency of
the selectivity under high pressure. The higher solubility of water
in hexane at higher temperatures is compensated due to the pres-
sure induced hydration of the enzyme, which finally leads to similar
selectivities within the examined temperature range.
4. Conclusion
In summary under each examined reaction conditions the
application of pressure leads to an increase in the activity of Can-
dida rugosa lipase. Regarding the investigated system, pressures
between 50 and 200 MPa lead to a slight decrease in the enzyme
activity at reaction temperatures of 20 ◦C and 35 ◦C. A higher sta-
bility and activity at higher temperatures combined with high
pressure could be observed.
Furthermore, a strong increase in the selectivity at pressures
between 50 and 200 MPa was observed, likewise under each exam-
ined reaction conditions. This is clearly different from the behavior
at ambient pressure where the lipase exhibits a strong dependency
of the eeR from the water content of the enzyme. This study leads
to the assumption that at high pressures the eeR mainly depends
on the packing state of the enzyme due to compression or is driven
by structural changes of the active center. Thus, it has been proven
that high pressure could be a useful tool to influence the transes-
terification, calalyzed by Candida rugosa lipase in organic solvents,
by increasing the specific activity as well as the selectivity.
3.3. Influence of water content under high pressure
The hydration level of the enzyme is strongly influenced by the
use of organic solvents as well by the application of pressure, as dis-
cussed above. In order to investigate the direct influence the water
content of the solvent and of the enzyme reactions were carried out
in untreated, anhydrous and in water-containing (1 ␮L water/mL
solvent) hexane.
As expected, the specific activity is enhanced with increasing
water content due to a higher flexibility of the enzyme caused by
the water molecules (Fig. 6(A)). This effect is still present at high
pressures, whereas the increase was higher when the reactions
were carried out under high pressure (0.1 MPa 1.7 fold; 50 MPa 2.1
fold; 100 MPa 2.3 fold; 200 MPa 2.2. fold, with respect to the spe-
cific activity exhibited in untreated, anhydrous hexane). Besides,
the reactions exhibit a similar profile with the highest conversion
at 50 MPa. A further increase of pressure leads to a slight decrease
in the activity. From these results it can be assumed that the above
Acknowledgements
The authors wish to thank the Deutsche Forschungsgemein-
schaft DFG for the financial support of the projects well as for one
of the authors (DH). The authors also thank Amano Enzymes Inc.
Japan for the generous gifts of the lipase.

110
D. Herbst et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 104–110
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