Stability improvement of immobilized Candida antarctica lipase B in an organic medium under microwave radiation

Article abstract of DOI:10.1039/b401145g

The influence of microwave heating on the stability of immobilized Candida antarctica lipase B was studied at 100°C in an organic medium. The microwave radiation was carried out before enzymatic reaction (storage conditions) or during the enzymatic catalysis (use conditions). In both cases, enzymatic stability was higher under microwave heating than under conventional thermal heating, in strictly identical operating conditions. Furthermore, the gain of enzymatic stability under microwave heating appears to be higher in a more polar solvent, which interacts strongly with the microwave field. Our results suggest that microwave radiation has an effect, not related to temperature, on the process of enzymatic inactivation.

Full text of DOI:10.1039/b401145g

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Stability improvement of immobilized Candida antarctica
lipase B in an organic medium under microwave radiation
Barbara Réjasse,* Sylvain Lamare, Marie-Dominique Legoy and Thierry Besson
Laboratoire de Biotechnologies et Chimie Bioorganique, CNRS FRE-2766,
UFR Sciences Fondamentales et Sciences pour l’Ingénieur, Bâtiment Marie Curie,
Université de la Rochelle, F-17042, La Rochelle cedex 1, France
Received 26th January 2004, Accepted 19th February 2004
First published as an Advance Article on the web 10th March 2004
The influence of microwave heating on the stability of immobilized Candida antarctica lipase B was studied at 100 ЊC
in an organic medium. The microwave radiation was carried out before enzymatic reaction (storage conditions)
or during the enzymatic catalysis (use conditions). In both cases, enzymatic stability was higher under microwave
heating than under conventional thermal heating, in strictly identical operating conditions. Furthermore, the gain of
enzymatic stability under microwave heating appears to be higher in a more polar solvent, which interacts strongly
with the microwave field. Our results suggest that microwave radiation has an effect, not related to temperature, on
the process of enzymatic inactivation.
Effects of microwave radiation on Novozym®435 stability
have been studied in an organic medium when radiation was
carried out before the reaction (storage conditions) or during
the enzymatic catalysis (use conditions). The results obtained
Introduction
Microwave radiation as an energy source to heat is today widely
used in organic chemistry. Indeed, an electromagnetic field of
high frequency (2.45 GHz) induces molecular rotation of di-
were compared with data observed under conventional thermal
polar species, which is accompanied by intermolecular friction
heating in strictly identical operating conditions.
and subsequent dissipation of energy by heating in the core.
Reductions in reaction times, enhancements in conversions and
sometimes in selectivity have been reported concerning espe-
Results
cially solvent-free reactions conducted under a microwave
Kinetics of the butyl butyrate synthesis
field.1a–c
In order to determine the kinetics of the alcoholysis model
reaction, typical butyl butyrate syntheses were performed by
adding different amounts of immobilized lipase to an equi-
molar mixture of substrates (ethyl butyrate/butanol, 11/11
mmol) at 100 ЊC. Experiments were carried out under con-
ventional and microwave heating to detect a possible effect of
the mode of heating on the reaction kinetic. Experiments were
carried out in similar vessels and without internal agitation to
be sure that differences observed were only consequent upon
heating type.
In enzymatic synthesis, the use of microwave radiation
remains limited. Literature on this subject is still poor and often
controversial. First studies carried out in aqueous solutions
with various enzymes did not demonstrate any effect of micro-
wave field on enzymatic activity and stability.2,3 More recently,
still in an aqueous medium, it was reported that inactivation of
a pectin methylesterase was faster in the microwave heating
mode than in the conventional thermal heating mode, suggest-
ing the presence of nonthermal effects under microwave
radiation.4 Similar results were obtained with thermophilic
enzymes, on which microwave radiation has induced protein
structural rearrangements not related to temperature.5 On the
other hand, kinetic and selectivity improvements of enzymatic
reactions carried out under a microwave field in organic media
have been reported,6–9 but effects of microwave radiation on
enzyme stability in non-conventional media have not been yet
investigated. The aim of the present work was to study the
stability of the Novozym®435 (immobilized preparation of the
Candida antarctica lipase B widely used in enzymatic synthesis
in non-conventional media) under microwave heating com-
pared to conventional heating.
The amount of butyl butyrate obtained after 15 min of
reaction with various quantities of Novozym®435 is shown in
Fig. 1. As expected, butyl butyrate production increases with
the amount of enzyme. The linear behaviour for small quanti-
The model reaction chosen to test enzymatic activity after
incubation at high temperature (100 ЊC) was an alcoholysis
between ethyl butyrate and butanol, conducted in equimolar
conditions (11/11 mmol) and without solvent addition (Scheme
1). In these conditions, the hydrolysis reaction (corresponding to
butyric acid appearance) was so negligible (<0.5%) that butyl
butyrate synthesis might be used to follow Novozym®435
activity.
Fig. 1 Effect of the amount of Novozym®435 used for the butyl
butyrate synthesis (᭺, conventional heating; ×, microwave heating).
Scheme 1 Scheme of the model reaction.
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ties of enzyme confirms that diffusion at 100 ЊC was sufficient
to obtain reproducible experiments without internal agitation.
For all immobilized lipase quantities tested, the same initial rate
was observed under conventional and microwave heating (in
our conditions, Novozym®435 has an activity of 6.3 µmol
minϪ1 mgϪ1 for both heating modes).
Butyl butyrate synthesis was conducted until equilibrium
with 10 mg (limiting quantity, linear part of the curve Fig. 1) or
25 mg of Novozym®435 under conventional and microwave
heating to determine the equilibrium constant of the reaction
(Fig. 2). For both enzyme quantities tested, the same equi-
librium constant (Keq = 2.25) was obtained under conventional
and microwave heating.
activity against 13% when it was preincubated in butanol.
Under microwave heating, different behaviour was observed:
after 30 min of preincubation in butanol or ethyl butyrate, the
same residual activity (45%) was obtained. So, Novozym®435
appears to be more stable under microwave heating than under
conventional heating in both organic substrates. These results
show a difference between conventional and microwave heat-
ing, modifying the enzymatic inactivation process. Contrary to
stability studies previously cited, the temperature rise, which is
normally different according to heating type, has no influence
in our results. Indeed, in our experiments, the temperature of
the preincubation medium was fixed at 100 ЊC before rapidly
adding the biocatalyst.
Control tests were also conducted to observe how
Novozym®435 may react when it was irradiated alone, without
organic substrate. In these conditions, the biocatalyst weakly
interacts with the microwave field: after 30 min radiation at 300
W, the temperature of the immobilized lipase did not exceed
80 ЊC and only 20% of the enzymatic activity was lost (under
conventional heating, Novozym®435 loses 17% of its activity
after 30 min of incubation at 100 ЊC without solvent).
Enzymatic inactivation kinetics in storage conditions
The enzyme deactivation constant (kd) has been calculated from
the slopes of the best-fit curves obtained by linear regression
when log (A/A0 × 100) was plotted against preincubation time
(Fig. 4), where A was residual enzymatic activity obtained after
preincubation and A0 was enzymatic activity obtained without
preincubation. The deactivation of Novozym®435 in butanol
or ethyl butyrate obeyed first-order deactivation kinetics, for
both heating modes, according to an irreversible deactivation
mechanism in one step.
Fig. 2 Reaction kinetics obtained under microwave heating compared
to that provided under conventional heating (᭺, conventional heating,
25 mg Novozym®435; ×, microwave heating, 25 mg Novozym®435;
∆, conventional heating, 10 mg Novozym®435; ϩ, microwave heating,
10 mg Novozym®435).
Butyl butyrate synthesis was conducted in the same
conditions with substrates and biocatalyst of which the
thermodynamic activity of water was fixed at 0 before reaction.
Identical enzymatic activity and equilibrium constant were
found again, for both heating types.
These results show that in our conditions, the heating mode
has no effect on the initial rate and conversion rate of the
reaction.
Enzymatic stability in storage conditions
In order to study the real impact of the mode of heating on the
enzymatic stability, 25 mg of Novozym®435 were preincubated
in butanol or ethyl butyrate at 100 ЊC for various times, under
conventional or microwave heating. Residual enzymatic activ-
ities obtained after preincubation are shown in Fig. 3. Under
conventional heating, as expected in an organic medium,10
enzymatic stability was found to be higher in ethyl butyrate,
which is less polar than butanol: after 30 min of preincubation
at 100 ЊC in ethyl butyrate, the enzyme maintains 39% of its
Fig.
4
Linear regression curves obtained from logarithmic
representation of residual activity versus preincubation time (᭺,
preincubation in butanol, conventional heating; ∆, preincubation in
ethyl butyrate, conventional heating; ×, preincubation in butanol,
microwave heating; ϩ, preincubation in ethyl butyrate, microwave
heating).
In addition (Table 1), we have calculated from the kd, the
half-life time (t1/2) and the free energy of activation (∆Gd) for
the deactivation process. The half-life time was the time
required for 50% loss of enzyme activity whereas ∆Gd was
calculated from the following equation:
∆Gd = ϪRTln [kdh/kBT ]
where kd is the deactivation constant (hϪ1), kB Boltzmann’s
constant (1.38 × 10Ϫ23 J KϪ1), h Planck’s constant (1.84 ×
10Ϫ37), R the gas constant (8.314 J molϪ1
K
Ϫ1) and T the
temperature (K).
As shown in Table 1, the deactivation constant (kd) in
butanol is 6 times higher under conventional heating than
under microwave heating, whereas the kd in ethyl butyrate is
nearly the same for both heating types. Similar results were
obtained when aw was not fixed at 0 before the experiment.
Fig. 3 Residual activity after preincubation in organic substrates (᭺,
preincubation in butanol, conventional heating; ∆, preincubation in
ethyl butyrate, conventional heating; ×, preincubation in butanol,
microwave heating; ϩ, preincubation in ethyl butyrate, microwave
heating).
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Table 1 Kinetic and thermodynamic parameters of Novozym®435 inactivation in storage conditions in organic substrates at 100 ЊC
Storage substrate
aw
Heating mode
kd (hϪ1
)
t1/2 (h)
∆Gd (kJ molϪ1
)
Butanol
Butanol
Ethyl butyrate
Ethyl butyrate
Butanol
Butanol
Ethyl butyrate
Ethyl butyrate
0
0
0
0
Conventional
Microwave
Conventional
Microwave
Conventional
Microwave
Conventional
Microwave
10.04
1.59
3.10
2.71
10.21
3.14
2.36
2.36
0.030
0.189
0.097
0.111
0.029
0.096
0.128
0.128
110.29
116.00
113.93
114.35
110.23
113.89
114.78
114.78
no fixed
no fixed
no fixed
no fixed
Enzymatic inactivation under microwave heating is faster in the
less polar substrate, ethyl butyrate. This result is in contrast
with data obtained under conventional heating.
Discussion
At high temperature, enzymatic inactivation (thermodenatur-
ation) consists of the loss of the catalytically active conform-
ation. The stability of this active conformation is closely
dependent on interactions between enzyme and its micro-
environment. Indeed, different non-covalent forces such as
hydrogen bonds, hydrophobic, ionic and Van der Waals inter-
actions maintain enzymatic structure. At high temperature,
these forces are reduced and enzyme molecules unfold. All
the processes causing thermal inactivation of enzymes require
water.11,12 So, in an organic medium, where hydrophobic inter-
actions are strongest, enzyme structural rigidity is increased,
resulting in greatly enhanced thermal stability.13 Consequently,
enzymatic stability depends on the nature of the solvent and is
higher in hydrophobic than in hydrophilic solvents.10
Enzymatic stability in use conditions
Biocatalysts are generally more stable in use than in storage
conditions, because substrate and product locations and inter-
actions in the catalytic site stabilize the native enzymatic con-
formation. This could explain why no effect of heating mode
has been observed when a typical synthesis was conducted at
100 ЊC without enzymatic preincubation in an organic medium
(Fig. 2). To confirm this hypothesis, 25 mg of Novozym®435
were re-used for six successive runs carried out under con-
ventional or microwave heating. The initial rate and conversion
rate at 6 h obtained for each run and each heating mode are
shown Figs. 5 and 6.
In our study, typical behaviour has been observed concerning
Novozym®435 stability under conventional heating: enzyme
deactivation constant (kd) is more than 3 times higher in
butanol than in ethyl butyrate which is more apolar.
On the other hand, surprising behaviour has been observed
under microwave radiation: the enzyme deactivation constant is
1.7 times higher in ethyl butyrate than in butanol although the
same residual enzymatic activity was obtained in both cases
after 30 min preincubation at 100 ЊC.
Furthermore, Novozym®435 stability was enhanced under
microwave heating compared to conventional heating, espe-
cially in the more polar solvent butanol: kd is more than 6 times
smaller, leading to a residual enzymatic activity 3 times greater
after 30 min of preincubation at 100 ЊC. Under microwave heat-
ing, the polarity of solvent is an important parameter: the more
polar the solvent is, the greater its ability to couple with the
microwave energy.1a,b,c Thus, a 24 W power is enough in butanol
to maintain the temperature at 100 ЊC although 210 W are
required in ethyl butyrate.
Fig. 5 Evolution of the initial rate with the number of biocatalyst
re-uses.
The reasons for the increased Novozym®435 stability under
microwave radiation are still unclear. Our results suggest that
the microwave field changes the interactions between the
enzyme and its microenvironment, preventing the enzyme
thermodenaturation.
Conclusions
Fig. 6 Evolution of the conversion rate at t = 6 h with the number of
biocatalyst re-uses.
The ability to re-use a biocatalyst is a decisive parameter for
the economic viability of a biocatalytic process. In this study,
we have shown that the stability of immobilized Candida
antarctica lipase in organic media could be enhanced by
using microwave dielectric heating rather than conventional
thermal heating. Furthermore, for the first time, the influence
of microwave radiation on enzymatic stability in an organic
medium has been studied and the existence of an effect of
microwave field on the enzymatic inactivation process has been
highlighted. The increase of enzymatic stability under a micro-
wave field could explain enhancements in conversion rate
observed for some enzymatic syntheses carried out under
microwave heating. Enzymatic behaviour under microwave
radiation is actually studied with various biocatalysts and
solvents.
For both heating types, the initial rate of the butyl butyrate
synthesis decreases progressively when the number of runs
increases (Fig. 5). As soon as the biocatalyst was re-used, the
initial rate was higher under microwave heating compared to
conventional heating. After 6 runs, a factor of 2 was reached
between the remaining activity of the irradiated and con-
ventionally heated biocatalyst. Concerning the conversion rate
after 6 h of reaction, the same phenomenon was observed (Fig.
6). From the first re-use of Novozym®435, the thermodynamic
equilibrium of the reaction was no longer reached for either
heating type, but the conversion rate was even more important
under microwave heating, until reaching a factor of 2 after 6
runs.
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One of the two substrates (11 mmol) was placed in the quartz
Experimental
vessel and heated to 100 ЊC under conventional or microwave
heating. When the temperature of the substrate solution was
stable, 25 mg of Novozym®435 were rapidly added and the
mixture was incubated for various times at 100 ЊC under con-
ventional or microwave heating (24 W in butanol, 210 W in
ethyl butyrate). After the preincubation at 100 ЊC, the second
substrate (11 mmol) was added to the mixture to test the resi-
dual activity of the lipase. This activity test was conducted for
3 min (initial rate conditions) under conventional heating in
each case (100 ЊC), to be sure that only the influence of the
heating mode on enzymatic stability was observed.
Enzymatic and chemical materials
Candida antarctica lipase B immobilized on macro-porous
polyacrylic resin beads (Novozym®435), water content 2%,
activity 7000 PLU gϪ1 was procured from Novozymes A/S
(Bagsvaerd, Denmark). Ethyl butyrate, butyl butyrate, butyric
acid were purchased from Sigma Chemical Co. (USA).
Butanol, ethanol and acetonitrile were obtained from Carlo
Erba Reagenti (Italy). All substrates and solvents were of the
highest purity (99% minimum).
Microwave equipment
Enzymatic stability in use conditions
Reactions were performed in a Synthewave S402 microwave
oven (300 W monomode system; Prolabo, France) equipped
with a variable speed rotation system and an infrared temper-
ature detector. The temperature of the reaction mixture was
controlled using an algorithm, which allows the temperature
to be set at a given value by varying the power between 20 and
200 W to operate under the electromagnetic field over the
reaction.14,15
Typical butyl butyrate syntheses were carried out at 100 ЊC with
25 mg Novozym®435 under conventional or microwave heating
for 6 h. After 6 h, the reaction medium was filtered in order to
recover the biocatalyst. The enzyme so recovered was rinsed by
2 ml ethyl butyrate under vacuum before being re-used in
another identical experiment.
Acknowledgements
Gas chromatographic analysis
B.R. thanks the Conseil Régional de Poitou-Charentes for a PhD
grant.
The GC analysis was performed with a Hewlett Packard model
5890A instrument equipped with flame ionization detector
(FID) and an OV 01 fused silica capillary column (Chrompack,
France). The split ratio was 68/1.2. Injector and detector were
kept at 220 and 250 ЊC respectively. Carrier gas was nitrogen
and the flow rate in the column was 1.2 ml minϪ1. Hydrogen
and air were supplied to the FID at 45 and 350 ml minϪ1
respectively.
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Enzymatic stability in storage conditions in each organic
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