Continuous green biocatalytic processes using ionic liquids and supercritical carbon dioxide

Article abstract of DOI:10.1039/b200055e

Soluble Candida antarctica lipase B dissolved in ionic liquids showed good synthetic activity, enantioselectivity and operational stability in supercritical carbon dioxide for both butyl butyrate synthesis and the kinetic resolution of 1-phenylethanol processes by transesterification.

Full text of DOI:10.1039/b200055e

Continuous green biocatalytic processes using ionic liquids and
supercritical carbon dioxide
a
a
b
b
a
Pedro Lozano, Teresa de Diego, Daniel Carrié, Michel Vaultier and José L. Iborra*
a
Departamento de Bioquímica y Biología Molecular B e Inmunología. Facultad de Química, Universidad
de Murcia, P.O. Box 4021, E-30100 Murcia, España. E-mail: jliborra@um.es; Fax: 34.968.36.41.48;
Tel: 34.968.36.73.98
b
Université de Rennes1, Institut de Chimie, UMR CNRS 6510, Campus de Beaulieu. Av. Général Leclerc,
3
6
5042 Rennes, France. E-mail: Michel.Vaultier@univ-rennes1.fr; Fax: 33.2 99 28 69 55; Tel: 33 2 99 28
2 74
Received (in Cambridge, UK) 4th January 2002, Accepted 14th February 2002
First published as an Advance Article on the web 4th March 2002
Soluble Candida antarctica lipase B dissolved in ionic liquids
showed good synthetic activity, enantioselectivity and op-
erational stability in supercritical carbon dioxide for both
butyl butyrate synthesis and the kinetic resolution of
was used as biocatalyst. Both the synthesis of butyl butyrate
from vinyl butyrate and butan-1-ol, and the kinetic resolution of
rac-1-phenylethanol by transesterification with vinyl propio-
nate were selected as reaction models. In all cases, the synthetic
activity of each enzyme-IL system was continuously tested by
1-phenylethanol processes by transesterification.
2
1
operation/storage cycles. During the operation period (4 h d
)
The use of enzymes in organic solvents rather than in aqueous
media greatly enhances their technological applications.1
However, volatile organic solvents (vos) have a detrimental
impact on the environment and human health, and it is necessary
to develop green and clean reaction media before scaling-up
biocatalytic processes of industrial interest. Neoteric solvents,
e.g. ionic liquids and supercritical fluids, seem to be promising
alternative because of their physical and chemical character-
an ISCO 220SX high pressure extraction apparatus was used as
a continuous packed bed reactor in the selected conditions (Fig.
2
1
1). The packed bed reactor was stored (20 h d ) under dry
conditions over P in a dessicator at room temperature.†
Table 1 summarizes the activity and stability parameters of
the CALB–[BMIM][Tf N] system for butyl butyrate synthesis
under different scCO conditions. As can be seen, lipase was
2 5
O
2
2
able to catalyse the transesterification reaction in all the assayed
conditions, the specific activity increasing with temperature; the
high selectivity ( > 95%) observed was due to the low water
content ( < 4% v/v) used, giving a conversion degree higher
than 50%. No activity was observed in the absence of enzyme.
The increased selectivity with temperature could be explained
by a reduction in water solubility in the ionic liquid fraction.
2
istics. Room temperature ionic liquids (ILs), have been shown
to be good solvents for many chemical3–5 and biochemical6
processes, and to have an exceptional ability to stabilize
enzymes during continuous operation.7 Supercritical CO
2
(
scCO
2
) has been widely described as an excellent solvent for
hydrophobic compounds, and is billed as clean technology for a
8
range of industrial extractive processes. However, scCO
2
has
However, the previously mentioned adverse effect of CO
also observed because the specific activity of the CALB–
[BMIM][Tf N] system with scCO was 10-fold lower than that
observed without scCO . The increase in synthetic activity
2
was
an adverse effect for enzymatic catalysis, because the enzymes
exhibit important deactivation phenomena probably due to local
2
2
7
pH changes caused by the CO
2
, or conformational changes
2
9
produced during the pressurization/depressurisation steps.
In this paper, we propose a new concept for continuous
biphasic biocatalysis, where a homogeneous enzyme solution is
immobilized in one liquid phase (working phase) and substrates
and products reside largely in a supercritical phase (extractive
phase). This concept is illustrated as follows: an aqueous
solution of Candida antarctica lipase B (CALB) dissolved in
Table 1 Activity and operational stability parameters of free Candida
antarctica lipase B dissolved in [BMIM][Tf
butyrate synthesis in scCO
2
N] for continuous butyl
2
CO
2
Specific
Half-life
Pressure/
Temp./°C MPa
density/g activity (U/ Selectivitya time
21
(cycles)b
mL
mg Enz
(%)
pure 1-ethyl-3-methylimidazolium triflimide ([EMIM][Tf
or 1-butyl-3-methylimidazolium triflimide ([BMIM][Tf
2
N])
N]),
2
40
15.0
12.5
15.0
0.792
0.618
0.336
44 ± 2.1
62 ± 3.8
71 ± 3.9
96 ± 0.8
98 ± 0.5
99 ± 0.9
284
101
12
5
0
1
00
a
Ratio between synthetic and acyl-donor consumption rates. b Cycles:
operation (4 h) followed by storage (20 h, 25 °C).
2
Fig. 2 Deactivation profiles of C. antarctica lipase B in [BMIM][Tf N] for
Fig. 1 A. Structure of 1-alkyl-3-methylimidazolium triflimide, obtained
according to Lozano et al. B. Experimental set-up.
2
continuous butyl butyrate synthesis in scCO under different conditions (5,
7
40 °C and 15.0 MPa; -, 50 °C and 12.5 MPa; :, 100 °C, 15.0 MPa).
6
92
CHEM. COMMUN., 2002, 692–693
This journal is © The Royal Society of Chemistry 2002

Table 2 Activity and operational stability parameters of free Candida antarctica lipase B dissolved in ionic liquids for continuous (R)-1-phenylethyl
2
propionate synthesis in scCO at 15 MPa
Specific activity (U/mg
Enz.)
Half-life time
(cycles)
Ionic liquid
Temp.°C
Selectivity (%)
Ee (%)
[
EMIM][Tf
2
N]
50
1.6 ± 0.3
1.1 ± 0.1
1.7 ± 0.2
0.6 ± 0.1
0.2 ± 0.02
86.3 ± 1.3
95.2 ± 1.5
84.8 ± 3.2
88.1 ± 4.6
81.5 ± 2.9
> 99.9
> 99.9
> 99.9
> 99.9
> 99.9
24
16
22
8
1
00
50
00
50
[
BMIM][Tf
2
N]
1
Nonea
10
a
Lyophilised powder of aqueous enzyme solution adsorbed on Celite.
with increasing temperature could also be related to a reduction
in scCO
density. Almeida et al.9 also reported enhanced
activity of immobilized CALB (Novozyme) with decreasing
scCO densities, which they attributed to a reduction in the
adverse effects produced by the enzyme solvation with CO
enzymatic biotransformations of industrial interest can be
carried out.
This work was partially supported by CICYT grant BIO99-
0492-C02-01. We thank Ms C. Sáez for technical assistance and
Novo España, S.A. for the gift of Novozym 525 solution.
2
2
2
6
molecules. Blanchard et al. reported that scCO
up 0.6 mole fraction for [BMIM][PF ]) in the IL phase, and so
a decrease in scCO density could improve the transfer-rate of
2
can dissolve
(
6
2
Notes and references
† Typical experimental procedure for butyl butyrate synthesis: the soluble
substrates to the enzyme microenvironment, thus favouring the
enzyme action. Fig. 2 depicts activity loss profiles obtained
when the CALB–[BMIM][Tf
2
N] system was reused in the
2
enzyme (0.6 mg/65 mL water) was dissolved in 2 mL [BMIM][Tf N], and
different supercritical conditions. As can be seen, activity decay
was enhanced by the increase in temperature. In all cases,
enzyme deactivation followed first-order kinetics. Table 1
shows the half-life time of the enzyme. The protective effect of
the IL against thermal and solvent denaturation was clearly
observed because the enzyme showed practically the same half-
then introduced into the 10 mL cartridge of an ISCO 220SX high pressure
extraction apparatus, containing 3 g of glass wool. Reactions were carried
out by continuously pumping substrate solution (0.38 M vinyl butyrate and
21
0
.76 M butan-1-ol in hexane) at 0.1 mL min , which was mixed with the
scCO
scCO
2
flow of the system. Substrates and products were fully soluble in
2
, and the reaction mixture was recovered by depressurising through a
21
calibrated heated restrictor (1 mL min , 70 °C) every 30 min. Samples
life time with and without scCO
2
. Additionally, the enzyme
were analysed by GC.7
exhibited interesting activity and stability levels at 100 °C, a
‡
Typical experimental procedure for kinetic resolution of rac-1-phenyl-
clearly denaturative condition in free scCO
2
reaction me-
ethanol. The soluble enzyme (1.3 mg/150 mL water) was dissolved in 4 mL
ionic liquid, and then introduced into the 10 mL cartridge of a ISCO 220SX
high pressure extraction apparatus, containing 3 g Celite. The enzyme
without ionic liquid was prepared by adsorption of the aqueous enzyme
solution (1.3 mg/4 mL) in the same amount of Celite, and then lyophilising.
Reactions were carried out by continuously pumping substrate solution (50
7
dium. ‡
The synthetic activity of two CALB-IL ([EMIM][Tf
N]) systems in scCO was also tested for an
2
N] and
[
BMIM][Tf
2
2
enantioselective reaction (see Table 2). As can be seen, all the
assayed conditions were able to catalyse the racemic resolution
of rac-1-phenylethanol. However, the synthetic activity of the
enzyme and selectivity of this reaction were lower than in the
case of butyl butyrate synthesis. These results were the
consequence of a drop in nucleophilic power from butan-1-ol to
rac-1-phenyltheanol due to the primary position of the hydroxy
group in the former and the secondary position in the latter.
Furthermore, the suitability of this reaction system operating in
a continuous way was demonstrated by the high enantioselectiv-
ity exhibited by the enzyme, because the (S)-1-phenylethyl
propionate isomer was never detected, and the (R)-1-phenyl-
ethyl propionate isomer was always obtained at a conversion
degree higher than 35%. Additionally, the half-life times of
these CALB-ILs systems for continuous reuse are shown in
Table 2, where it can be seen that they are lower than those
observed for butyl butyrate synthesis. Furthermore, it seems that
the increase in polarity of IL caused by shortening the alkyl
chain on the N3 of the imidazolium ring slightly enhances all the
activity and operational stability parameters, probably due to a
more adequate microenvironment for the enzyme. Once again,
the protective effect of ILs towards enzyme deactivation by
mM vinyl propionate and 100 mM rac-1-phenylethanol in hexane) at 0.1
21
2
mL min , and mixed with the scCO flow of the system. Samples,
containing 10 mM butyl butyrate as internal standard, were analysed by GC
using a capillary Beta DEX-120 column (30 m 3 0.25 mm 3 0.25 mm,
Supelco) and a FID, under the following conditions: carrier gas (He) at 1
21
MPa (205 ml min total flow); temperature programme: 60 °C, 10 °C
21
min , 130 °C; split ratio, 100+1; detector, 300 °C. Other details are
included in the above protocol.
One unit of activity was defined as the amount of enzyme that produced
one micromole of product per min.
1
2
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In conclusion, this work clearly demonstrates the exciting
2
potential of combining ILs with scCO for carrying out
8
M. Perrut and E. Revenchon, in Proc. 7th Meeting on Supercritical
Fluids. Natural products processing, Vol. 2, ed. M. Perrut and E.
Revenchon, Vandeouvre, I.N.P.L., 2000.
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provide the enzyme with an adequate microenvironment,
allowing high activity, enantioselectivity and stability, together
9
S. Kamat, J. Barera, . J. Beckman and A. J. Russel, Biotechnol. Bioeng.,
2
with their possible continuous reuse. Supercritical CO is seen
1
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easy product recovery. This paper shows how adequately
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