Bioreactors based on monolith-supported ionic liquid phase for enzyme catalysis in supercritical carbon dioxide

Article abstract of DOI:10.1002/adsc.200600554

Bioreactors with covalently supported ionic liquid phases (SILP) were prepared as polymeric monoliths based on styrene-divinylbenzene or 2-hydroxyethyl methacrylate-ethylene dimethacrylate, and with imidazolium units loadings ranging from 54.7 to 39.8 % wt IL per gram of polymer. The SILPs were able to absorb Candida antarctica lipase B (CALB), leading to highly efficient and robust heterogeneous biocatalysts. The bioreactors were prepared as macroporous monolithic mini-flow systems and tested for the continuous flow synthesis of citronellyl propionate in supercritical carbon dioxide (scCO ₂) by transesterification. The catalytic activity of these mini-flow-bioreactors remained practically nchanged for seven operational cycles of 5 h each in different supercritical conditions. The best results were obtained when the most hydrophobic monolith, M-SILP-8-CALB, was assayed at 80°C and 10 MPa, reaching a total turnover number (TON) of 35.8 × 10⁴ mol product/mol enzyme. The results substantially exceeded those obtained for packed-bed reactors with supported silica-CALB-Si-4 catalyst under the same experimental conditions.

Full text of DOI:10.1002/adsc.200600554

FULL PAPERS
DOI: 10.1002/adsc.200600554
Bioreactors Based on Monolith-Supported Ionic Liquid Phase for
Enzyme Catalysis in Supercritical Carbon Dioxide
Pedro Lozano,a Eduardo García-Verdugo,b Rungtiwa Piamtongkam,a,c
b
b,
Naima Karbass,b Teresa De Diego,a M.Isabel Burguete, Santiago V.Luis, *
a,
and JosØ L.Iborra *
a
Departamento de Bioquímica y Biología Molecular B e Inmunología, Facultad de Química, Universidad de Murcia, P.O.
Box 4021, 30100 Murcia, Spain
Phone: (+34)-9-6836-739; Fax: (+34)-9-6836-4148; e-mail: jliborra@um.es
Departamento de Química Inorgµnica y Orgµnica, UAMOA, Universidad Jaume I, Campus del Riu Sec, Avenida Sos
b
Baynant s/n, 12071 Castellón, Spain
Phone: (+34)-9-6472-8239; Fax: (+34)-9-6472-8214; e-mail: luiss@qio.uji.es
Program in Biotechnology, Faculty of Science, Chulalongkorn University, 10330-Bangkok, Thailand
c
Received: October 26, 2006; Revised: February 23, 2007
Abstract: Bioreactors with covalently supported unchanged for seven operational cycles of 5 h each
ionic liquid phases (SILP) were prepared as polymer- in different supercritical conditions.The best results
ic monoliths based on styrene–divinylbenzene or 2- were obtained when the most hydrophobic monolith,
hydroxyethyl methacrylate–ethylene dimethacrylate, M-SILP-8-CALB, was assayed at 808C and 10MPa,
and with imidazolium units loadings ranging from reaching a total turnover number (TON) of 35.8”104
54.7 to 39.8% wt IL per gram of polymer. The SILPs mol product/mol enzyme.The results substantially
were able to absorb Candida antarctica lipase B exceeded those obtained for packed-bed reactors
(CALB), leading to highly efficient and robust heter- with supported silica-CALB-Si-4 catalyst under the
ogeneous biocatalysts.The bioreactors were pre-
pared as macroporous monolithic mini-flow systems
and tested for the continuous flow synthesis of citro-
same experimental conditions.
nellyl propionate in supercritical carbon dioxide Keywords: continuous processes; enzyme catalysis;
(scCO2) by transesterification.The catalytic activity green chemistry; supercritical fluids; supported ionic
of these mini-flow-bioreactors remained practically liquids
Introduction
catalytic processes.[4,5] Several strategies have been de-
veloped to stabilise the enzyme (e.g., covalent attach-
Supercritical carbon dioxide (scCO2) is an excellent ment on supports coated with hydrophilic polymers,
ꢀgreenꢁ solvent with a huge potential for both the syn- entrapment in silica-aerogels, cross-linking enzyme
thesis and separation of chemicals.It represents an in-
aggregates, etc.).[6,7] However, the best results for
teresting alternative to organic solvents for develop- enzyme catalysis in scCO2 have been obtained when
ing cleaner and more environmentally friendly chemi- ionic liquids (ILs) were applied as liquid immobilisa-
cal processes, because it provides a clean, non-toxic, tion supports for the enzymes.[8] In this context, bio-
non-flammable and tuneable solvent system, which is catalytic IL/scCO2 biphasic systems, based on en-
easily removed to leave reaction products free from zymes retained into the IL phase (catalytic phase),
undesirable organic residues.[1] The greenness of while substrate and product reside largely in the
chemical transformations in scCO2 should also con- scCO2 (extractive phase), were the first attractive and
cern the applied catalyst.[2] Thus, one of the most efficient approach for continuous green processes.[8a,b]
straightforward “green” practical applications of The advantages to use of ILs as additives or coating
scCO2 is its combined use with enzymes.Since the
pioneering work of Randolph et al.and Hammond
material to enhance lipase-catalysed transesterifica-
tion in either organic solvents or supercritical fluids
et al.[3] in 1985, scCO2 has been described as a solvent have also been described.[9] In this way, ILs have
with a strong deactivating effect on enzymes, which is emerged as exceptionally interesting non-aqueous re-
the main drawback for application in industrial bio- action media for enzymatic transformations mainly
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due to their ability to maintain a high level of activity,
stereoselectivity, and stability of enzymes in chemical
transformations,[10] even under extremely harsh condi-
tions (e.g., scCO2 at 1508C and 100 bar).[11] The physi-
cal properties of ILs (density, viscosity, melting point,
polarity, etc) can be finely tuned by appropriate struc-
ture selection.Their interest as green solvents resides
in their high thermal stability and very low vapour
pressure, which mitigates the problem of the emission
of volatile organic solvents to the atmosphere.[12]
Although ILs have become commercially available,
they are still relatively expensive compared with tra-
ditional solvents.Besides, some of them show evi-
dence of low biodegradability and (eco)toxicological
properties.[13] Hence, the immobilisation of ILs onto a
support or structured material (e.g., by simple im-
pregnation, covalent linking of the cation, sol-gel
method, etc.) is a highly attractive strategy to mini-
mise the amount of ILs used, while maintaining their
catalytic properties.Additionally, supported ILs have
the advantage of easy separation and recyclability as
well as a potential for use in the development of con-
tinuous processes.[14]
Figure 1. Set-up of the reactor with immobilized CALB
onto monolith-supported ionic liquid phase for continuous
operation under flow conditions in scCO2.
Recently, we reported the preparation of supported
ionic liquid phases (SILP) as monolithic rods by the
surface modification of macroporous polystyrene–di-
vinylbenzene (PS-DVB) resins.[15] As this type of ma-
terial is not able to adsorb ILs as films onto the sur-
face, unlike silica gel,[14] our approach was based on
immobilisation by the covalent binding of IL-like
units (alkylimidazolium cations) to the polymer sur-
face.In this way, IL properties are transferred to the
solid phase, leading to a monolith-supported ionic
liquid phase (M-SILP) able to be used to develop
mini-flow reactors for continuous processes, which
offer many potential advantages from an industrial
point of view over existing batch techniques or
packed-bed reactors containing particulate cata-
lysts.[16]
Scheme 1. Kinetic mechanism for CALB-catalysed citronell-
yl propionate (3) synthesis from vinyl propionate (1) and cit-
ronellol (2) by transesterification.
In this paper, we develop a new generation of
highly efficient enzymatic reactors with suitable me-
chanical stability for continuous-flow processes in
scCO2 (Figure 1).The catalytic system is based on an
enzyme (Candida antarctica lipase B, CALB) immobi- Results and Discussion
lised by non-covalent attachment onto macroporous
M-SILPs, which were applied for a continuous trans- Synthesis and Characterisation of the Monolith-
esterification process in scCO2.The synthesis of citro-
Supported Ionic-liquid Phase (M-SILP)
nellyl propionate (3) from vinyl propionate (1) and
citronellol (2) catalysed by CALB was selected as re- The preparation of the polymeric monolithic materi-
action model (Scheme 1).This approach combines the als is depicted in Scheme 2.Monolithic polymer (M-
advantages of supported ionic liquid as a “supported 7) was synthesised by thermally induced radical solu-
liquid solvent”, those of an immobilised enzyme as a tion polymerisation of a monomeric mixture of p-
“green” catalyst and the use of a supercritical fluid as chloromethylstyrene (5) and divinylbenzene (6), using
a “green” reaction/extraction solvent with the advan- toluene/1-dodecanol as the precipitating porogenic
tages of a continuous flow process, easy product sepa- mixture and AIBN as the radical initiator (Table 1).
ration and catalyst reuse.
In order to take full advantage of the potential of
these monolithic systems, polymerisation was carried
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units per gram of polymer, which it is equivalent to
54.7% of IL by weight of polymer (Table 1). In order
to check if the microenvironment of the monolith was
modified by attachment of the IL-like units, the shift
experienced by a solvatochromic fluorescent probe, in
this case pyrene, was studied.[18] The pyrene value II /
IIII measured for M-SILP-8 clearly indicated a com-
pletely different microenvironment sensed by excited-
state pyrene.The pyrene I /IIII value was 1.44 for M-
I
SILP-8, which is significantly higher than that mea-
sured for M-7 (II /IIII =1.01) and similar to that ob-
served in methanol (II /IIII =1.33).[18]
Since these macroporous polymers are designed to
be used in the flow-through mode, they should be ma-
terials with a low flow resistance.Figure 3 shows that
for polymer M-SILP-8 the back pressure (normalised
to a length of 1 cm) was lower than 0.2MPacmÀ1 for
different flow rates (0.1–3.5 mL/min of CO2), temper-
atures and pressures, confirming the good pressure re-
sistance of this material to work as a packed-bed reac-
tor under scCO2.The low pressure drop observed
with the increase in flow can be ascribed to the for-
Scheme 2. Synthesis of monolith-supported ionic liquid
phase M-SILP-8 and M-SILP-12, i) 30% (w/w) of a mixture
5:6 (40:60 by weight) in a porogen mixture (75:25 dodeca-
nol:toluene by weight) 1% AIBN, 708C, 24 h.ii) butylimida-
zole, 808C.iii) 40% (w/w) of a mixture 9:10:11 (40:40:20 by
mol) in DEG, 1% AIBN, 708C, 24 h.
out within a stainless steel cartridge, which can be mation of monolithic polymers with a very high pore
adapted to our supercritical reactor allowing us to use size, as revealed by the SEM image (showing pores
it as a mini-reactor (1 mL volume) under flow condi- larger than 1 mm, Figure 3).The resulting monolithic
tions (Figure 1).The monolithic-supported ionic
columns had similar structural properties to those
liquid phase (M-SILP-8) was obtained by the post al- that have been used for chromatographic applications
kylation of M-7 with N-butylimidazole.The introduc-
and for other catalytic applications.[19,20]
tion of the imidazolium IL-like moieties was moni-
Polymer M-SILP-12 was synthesised by polymeri-
À
tored by the disappearance of the CH2 Cl group, sation of the corresponding monomeric ionic liquid
using both the FT-IR–Raman and a colorimetric [1-(p-vinylbenzyl)-3-methylimidazolium chloride, 9][21]
method based on the NBP test.[17] A 95% conversion with ethylene dimethacrylate (EDMA, 10) and 2-hy-
of the chloride groups was achieved after 5 h of reac- droxyethyl methacrylate (HEMA, 11) and using DEG
tion.Longer reaction times did not improve the con-
as poronic agent.
version degree (Figure 2).Most likely, a small per-
centage of the CH2 Cl groups are located in non-ac- was immobilised by simple adsorption of an aqueous
cessible highly cross-linked regions.The FT-Raman of
The enzyme [Candida antarctica lipase B (CALB)]
À
solution of CALB.The resulting enzymatic catalysts
M-SILP-8 showed bands characteristic of 1-butyl-3-p- (M-SILP-8-CALB and M-SLIP-12-CALB) showed a
methylstyrene–imidazolium chloride (1505, 1345, 1078 very high ionic liquid/enzyme ratio, which ensured
and 1029 cmÀ1, Figure 2).Elemental analysis showed
full interaction between the protein molecule and the
the loading of IL to be 1.98 mequivs. of imidazolium IL phase, and could favour enzyme stabilisation
Table 1. Characteristics of monolithic columns prepared according Scheme 2.
Entry
Monomeric composition (% wt.)[c]
ILs loading
Enzyme loading
IL/E
ratio
[d]
[e]
5
6
9
10
11
mequiv.IL/g supp.
g IL/g supp.(%)
mmol E/g supp.[f]
1[a]
2[b]
40
–
60
–
–
40
–
40
–
20
1.98
1.70
54.7
39.8
0.31
0.31
6387
5484
[a]
The porogenic mixture was toluene/1-dodecanol (1:3 w/w) with a 70:30 (w/w) porogens/monomers ratio.
The porogen used was DEG mixed with monomers in a 60:40 (w/w) porogen/monomer ratio.
The polymerisation was performed at 708C by using 1% AIBN.
[b]
[c]
[d]
[e]
Determined by elemental analysis.
Calculated as percentage of IL moieties per gram of support, considering 1-butyl-3-p-methylstyrene-imidozolium chloride
(Mw=276.14 gmolÀ1) as the IL moiety for M-SILP-8, and 1-methyl-3-p-methylstyrene–imidozolium chloride (Mw=
234.09 gmolÀ1) the IL moiety for M-SILP-12, respectively.
[f]
Mw for CALB was 33 kDa[4e]
.
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À
Figure 2. A: Synthesis of monolith-supported ionic liquid phase. B: Profile of substitution of CH2 Cl groups by butylimida-
zole units with time, following the disappearance of the CH2 Cl characteristic band at 1265 cm of the FT-Raman spectra.
À1
À
(Inset shows an expansion in the 1300–1200 cmÀ1 region for the FT-Raman spectra of M-7 at different reaction times). C:
FT-Raman spectra expansion from 1600–500 cmÀ1 for M-7 and M-SILP-8.
Figure 3. a) Micrograph of M-SILP-8 obtained by scanning electron microscopy. b) Pressure drop (DP, MPa cm À1)·for M-
À1
^
SILP-8 (normalised to a length of 1 cm) vs. CO2 flow rate (mLmin ) [( ) 408C, 8MPa, 1=0.311 g/mL. (&) 1008C, 10MPa,
~
1=0.191 g/mL. (”) 1008C, 15 MPa, 1=0.336 g/mL. ( ) 1008C, 8MPa, 1=0.143 g/mL.]
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(Table 1).[22] M-SILPs not only provide high flow char- under dry conditions) for two days, in order to ob-
acteristics and mechanical stability, based on the in- serve both the level of activity, as well as any deacti-
trinsic properties of these macroporous materials, but vation process that might take place.
also an easy and simple protocol to anchor the
It can be seen that, for all the immobilised deriva-
enzyme onto a modified matrix with “enzyme friend- tives, productivity in the synthetic process increased
ly” moieties like alkyl-imidazolium cations.[23]
with temperature, reaching a maximum level at 808C,
decreasing at 1008C.Temperature seems to be an ac-
tivating parameter for enzymatic catalysis, although,
in the case of scCO2, a decrease in the fluid density
has also been described as a positive parameter in
enzyme activity (see Table 2).[6,7,24] In this regard, the
activation of a commercial immobilised CALB prepa-
Synthesis of 3 by CALB-Monolithic Catalytic
Bioreactors in scCO2 under Continuous Flow
Conditions
The ability of each monolith-SILP-CALB derivative ration (Novozym 435) by temperature in different su-
to catalyse transesterification reactions in scCO2 was percritical fluids (ethane, propane and CO2) for ester
tested by using the synthesis of 3 as the activity test synthesis has also been described.[22a] This effect was
(see Scheme 1, Figure 1).Terpene esters, such as 3, directly related with the decrease in fluid density,
are very important flavour and fragrance substances, which produced a reduction in the internal diffusion
and they may be considered natural when produced limitations within the enzyme particle.The increase in
enzymatically.[24] For comparison, another CALB im- activity of lipase-catalysed acrylate synthesis in differ-
mobilised derivative, obtained by the adsorption of ent supercritical fluids (SF6, propane, ethane, ethyl-
the enzyme onto a modified silica gel with butyl side ene, HCF3 and CO2) under the same conditions
chains (Si-C4) was also studied.
(458C, 11MPa) was also associated with a decrease in
Figure 4 depicts all the productivity profiles for the fluid density.[24a] However, it is necessary to point out
synthetic product 3 catalysed by each immobilised de- that, in all the assayed conditions, the use of M-SILP
rivative obtained in continuous operation under dif- for enzyme immobilisation clearly improved produc-
ferent supercritical conditions (see Table 2).For each
tivity compared with CALB supported on silica-C4.
assayed condition, the catalyst operated in daily Thus, at 808C and 100MPa, the productivity of M-
cycles (5 h/d of synthetic process and 19 h of storage SILP-8-CALB was 4.5- and 7-times higher than that
obtained for M-SILP-12-CALB and silica-C4 deriva-
tives, respectively, reaching a total turnover number
(TON) of 35.8”104 mol product/mol enzyme.Further-
more, all derivatives showed excellent operational be-
haviour, with practically no loss of activity during the
assayed time, except in the case of the process carried
out at 1008C.The reaction was also very selective as
the enzyme-catalysed reaction was fully directed to-
wards the synthesis of 3, due to the absence of free
water molecules in the reaction system during the op-
eration time.Only during the initial stages was the
presence of the product of hydrolysis 4 observed for
all derivatives (see Table 2).The yield of this product
was extremely low and was not observed afterwards,
probably because all the free residual water molecules
remaining in the support after the immobilisation pro-
cess were consumed.[8,11]
Different control experiments showed, first, that
neither M-SILP-8 nor M-SILP-12 monoliths led to 3
Figure 4. Productivity profiles for synthesis of 3 catalyzed by
~
*
M-SILP-8-CALB ( ), M-SILP-12-CALB ( ) and silica-
&
butyl-CALB ( ) with operation time, in a continuous pro-
cess in the different assayed conditions (see Table 2 for ex-
perimental conditions A, B, C and D).
Table 2. Experimental assayed conditions and product yields obtained for M-SILP-8-CALB-catalysed synthesis of 3 in
scCO2.
Entry
Temp.Pressure
CO
2 density
[g/mL]
Inlet mass flow
[mmol/min]
Operation time
[h]
Yield [%]
[8C]
[MPa]
U
A
3
4
A
B
C
D
40
60
80
100
10
10
10
10
0.627
0.301
0.226
0.191
10
20
40
20
10
10
10
5
55.7Æ2.6
86.2Æ2.0
92.8Æ3.2
24.3Æ3.8
1.6Æ0.2
<0.1
<0.1
<0.1
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in the absence of CALB and, second, that no enzyme Experimental Section
leaching occurred, as demonstrated by the lack of ac-
Safety Warning: Experiments using large amounts of com-
pressed gases such as supercritical fluids are potentially haz-
ardous and must only be carried out using appropriate
equipment and safety precautions.
tivity at the exit of the system.In fact, it was necessa-
ry to wash each monolith-CALB derivative with
100 mL of an aqueous solution of acetonitrile (50%
v/v) to totally eliminate the adsorbed enzyme mole-
cules.This revealed the possibility of washing out the
deactivated enzyme and reactivating the reactor with
fresh enzyme using simple protocols.[26]
Materials
Candida antarctica lipase B (Novozym 525 L, EC 3.1.1.3)
was from Novozymes S.A. (DK). The enzyme solution was
These results should be analysed as a function of
both the mass transport of the substrates to the active ultrafiltered to eliminate all the low molecular weight addi-
tives, as follows: 25 mL Novozym 525 solution were diluted
in 225 mL water, and the resulting solution was concentrat-
ed 10-fold by ultrafiltration at 48C, using a Minitan (Milli-
pore) system equipped with polysulfone membranes
(5,000 Da cut-off).This process was repeated 3 times, result-
ing in a CALB solution of 13.26 mg/mL, as determined by
the Lowry method, and a purification degree of 1.6-fold.
Modified silica gel (32–63 mm particle size, 6 nm pore diame-
ter), containing butyl (Si-C4) groups was obtained from Ap-
plied Separations Inc (Allentown, PA, USA).Molecular
sites, and in the light of the different ability of each
support to provide the enzyme with an appropriate
microenvironment for catalysis.For example, the
large pore distribution of monoliths (e.g., see Fig-
ure 3A for M-SILP-8), together with the high effi-
ciency of scCO2 to transport hydrophobic compounds,
ensures very fast mass transfer directly through the
pores.[7,23,25] On the other hand, ILs have been de-
scribed as liquid immobilisation supports because
multipoint enzyme-IL interactions (ionic, hydrogen sieve UOP Type 4 (pore diameter 0.4 nm), substrates, sol-
vents and other chemicals were purchased from Sigma–Al-
drich-Fluka Co (Madrid, Spain), and were of the highest
purity available.
bonds, van der Waals, etc) may occur, resulting in a
supramolecular network able to maintain the protein
conformation active.[8,11,12] In this case, monoliths con-
taining covalently attached ILs not only provide an
adequate microenvironment for lipase action, but also
improve the mass-transfer phenomena of hydrophobic
substrates and products from the scCO2 phase, lead-
ing to a highly selective and stable immobilised
enzyme even at high temperatures and pressures.The
higher hydrophilicity and, probably, the different mor-
phologies leading to higher back pressures in the case
of M-SILP-12 resulted in a clear decrease in the
CALBꢁs synthetic activity.Similarly, a clearly hydro-
philic support, like silica gel, displayed the worst ac-
tivity level for the whole temperature range under
study.
Preparation of Monoliths
Monoliths 8 or 12 were molded into a stainless-steel car-
tridge (13”6.2 mm) of the ISCO 220SX high-pressure ex-
traction apparatus.M-7 was prepared using a solution of
0.01 g of azobisisobutyronitrile (AIBN) in p-chloromethyl-
styrene (0.4 g), divinylbenzene (80% w/w, 0.6 g), toluene
(0.58 g), and 1-dodecanol (1.75 g). The polymerisation mix-
ture was stirred and purged with nitrogen for 3 min and
poured into a mold.The stainless steel tubular mold was
sealed at the two ends, and placed in a vertical position in a
water bath.The polymerisation was allowed to proceed for
24 h at 708C.The seals were then removed, the tube was
provided with fittings, attached to a high pressure pump,
and THF was pumped through the column at a flow rate of
1 mLminÀ1 to remove the porogenic solvents and any other
soluble compounds.
Conclusions
Polymer M-12 was prepared with similar preparation pro-
tocols using the polymerisation mixture show in Table 1.
We have developed new monoliths-supported ionic
liquid phases (M-SILPs) with covalently attached IL-
like moieties that transfer the desired properties of
the liquid to the solid polymer.These M-SILPs can
be used as highly efficient supports for the non-cova-
lent immobilisation of enzymes and the preparation
of bioreactors for lipase-catalysed ester synthesis in
scCO2.The efficiency of the system was dependent on
both the microenvironment provided by the support
and the supercritical conditions, which are involved in
the mass-transfer phenomena.The methodology here
reported represents a simple and straightforward
strategy to improve the efficiency of an enzymatic re-
action under non-classical conditions, allowing the de-
velopment of green synthetic chemical processes.
Enzyme Immobilisation
Immobilised enzyme derivatives were prepared by simple
adsorption of an aqueous solution of CALB (2 mg dissolved
in 100 mL water) into monoliths, or silica-C4 (200 mg, previ-
ously packed in a stainless-steel cartridge).Then, each wet
support was stored under controlled Aw (0.11) conditions
over LiCl in a desiccator for 48 h at room temperature prior
to use.
Drying of Substrates
Water was removed from 1 and 2 by adding molecular
sieves (0.1 g/mL), shaking the resulting mixture for 24 h at
room temperature, and finally storing them in the presence
of the adsorbent.
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Sampaio, S.Barreiros, Biotechnol. Bioeng. 1996, 49,
399–404; d) N.Mase, T.Sako, Y.Horikawa, K.Takabe,
Tetrahedron Lett. 2003, 44, 5175–5178.
Synthesis of 3 in scCO2
A cartridge containig CALB-8, CALB-12 or CALB-Si-C4
derivative was placed into the ISCO 220SX system.The syn-
thesis of 3 was carried out by continuous pumping of a sub-
strate solution (1M of 1 and 2, respectively, in hexane) at
0.01–0.04 mL/min with an HPLC pump, and mixed with the
scCO2 flow of the system at 40–1008C and 10MPa (see
Figure 1).The reactor was continuously operated for 5 h/d,
followed by 19 h/d of storage into the ISCO system at room
temperature.The reaction mixture was recovered by contin-
uous depressurising through a calibrated heated restrictor
(1 mL/min, 408C) for 30 min steps.Samples were analysed
by GC.In all cases, the mass-balances of substrates and
products from the outlet were consistent with the substrates
mass-flow inlet.
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Analyses were performed with a Shimadzu GC-2010 instru-
ment equipped with FID detector.Samples were analysed
by a Beta DEX-225 column (30 m”0.25 mm”0.25 mm, Su-
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total flow); temperature program: 608C, 10 8C/min, 1808C;
split ratio, 50:1; detector, 3008C.
Acknowledgements
We would like to acknowledge the M.E.C (Refs.: CTQ2005–
01571 and CTQ 2005–08016), SENECA Foundation (Ref.:
02910/PI/05), BIOCARM(Ref.: BIO-BMC 06/01–0002) and
Bancaixa-UJI (Ref.: P1B2004–13) for the financial support.
E. G.-V. thanks the Ramón y Cajal Program (MEC) for per-
sonal financial support. We also thank Mr. Ramiro Martínez
from Novozymes EspaÇa, S.A. for the gift of enzymes
2002, 2,147–151; b) S.Park, RJ..Kazlauskas,
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