Microemulsion-based organogels as an efficient support for lipase-catalyzed reactions under continuous-flow conditions

Article abstract of DOI:10.1021/op500136c

In this work, microemulsion-based organogels (MBGs) containing lipase are reported as an efficient catalytic system for monoacylglyceride (MAG) synthesis. More specifically, Candida antarctica lipase B (CaLB) was immobilized on an MBG matrix (MBG_CaLB) formed with (hydroxypropyl)methyl cellulose as a gelling agent, and this catalyst revealed high conversion yields (expressed in grams of ester per hour per gram of enzyme) under both batch and continuous-flow conditions. Under flow conditions, 1:1 stearic acid/solketal (100 mM in n-heptane) could be converted to protected MAG (>99%) in a residence time of 11 min at 45 °C. A recycle study showed that the MBG_CaLB system can be recycled 15 times without activity loss, a number 2 times higher than under batch conditions catalyzed by immobilized lipase, in agreement with green chemistry protocols.

Full text of DOI:10.1021/op500136c

Article
pubs.acs.org/OPRD
Microemulsion-Based Organogels as an Efficient Support for Lipase-
Catalyzed Reactions under Continuous-Flow Conditions
Ivaldo Itabaiana, Jr,†,‡ Karen M. Goncalves,†,‡ Maria Zoumpanioti,§ Ivana C. R. Leal,‡
̧
Leandro S. M. e Miranda,† Aristotelis Xenakis,§ and Rodrigo O. M. A. de Souza*,†
†Biocatalysis and Organic Synthesis Group, Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
CEP22941909
‡Pharmacy School, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil CEP22941909
§Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 11635 Athens, Greece
ABSTRACT: In this work, microemulsion-based organogels (MBGs) containing lipase are reported as an efficient catalytic
system for monoacylglyceride (MAG) synthesis. More specifically, Candida antarctica lipase B (CaLB) was immobilized on an
MBG matrix (MBGCaLB) formed with (hydroxypropyl)methyl cellulose as a gelling agent, and this catalyst revealed high
conversion yields (expressed in grams of ester per hour per gram of enzyme) under both batch and continuous-flow conditions.
Under flow conditions, 1:1 stearic acid/solketal (100 mM in n-heptane) could be converted to protected MAG (>99%) in a
residence time of 11 min at 45 °C. A recycle study showed that the MBGCaLB system can be recycled 15 times without activity
loss, a number 2 times higher than under batch conditions catalyzed by immobilized lipase, in agreement with green chemistry
protocols.
For industrial purposes, continuous-flow systems are
preferred to batch reactors because they provide greater
process control, higher productivity, and improvement of
quality/purity and yield.14,15 Several types of reactor can be
used in continuous operation, among which packed-bed
reactors (PBRs) are the most popular because of their high
efficiency, low cost, and ease of construction, operation, and
maintenance.16,17
In our continuous work on the development of more
efficient biocatalytic systems,3 here we report our results on the
use of MBGs containing lipase under batch and continuous-
flow conditions applied to the synthesis of various protected
monoacylglycerol esters.
1. INTRODUCTION
Reverse micelles, or water-in-oil (w/o) microemulsions, have
already been reported as an effective medium for enzyme
immobilization,1,2 as they have been successfully used in several
cases to improve reaction yields using lower amounts of
biocatalyst and to enhance enzymatic activity compared with
conventional systems.3−6 Reverse micelles are usually defined
as spherical aqueous nanodroplets capped with a monolayer of
surfactant and/or cosurfactant molecules and isotropically
distributed within an oil.7
These structures form a thermodynamically stable and
optically transparent liquid medium with large interfacial area
that provides an aqueous domain where hydrophilic enzymes
can be hosted, an interface where the active site of enzymes can
be anchored, and a nonpolar organic phase where the
hydrophobic substrates or products may be dissolved.8,9
However, the main drawback of these systems is the difficulty
in isolating the products due to the presence of surfactants that
hinder phase separation.
Nevertheless, some microemulsions can be transformed to
gels by adding a gelling agent, usually a biopolymer such as
gelatin, agar, or a cellulose derivative,10 to form so-called
microemulsion-based organogels (MBGs). MBGs are rigid and
stable in various nonpolar or relatively polar organic solvents
and therefore can be used for several biotransformations in
organic media, such as hydrolysis, esterification, and other
syntheses.11,12 The gel matrix formed by the gelling agent, such
as a cellulose derivative, fully retains the surfactant, water, and
enzyme components and can be handled as an immobilized
biocatalyst that facilitates the diffusion of nonpolar substrates
and products.13 The network of the gel is considered to contain
a bicontinuous phase that may coexist with conventional w/o
microemulsion droplets containing the encapsulated enzyme.10
2. MATERIALS AND METHODS
2.1. Materials. Lipase B from Candida antarctica (CaLB),
sodium bis(2-ethylhexyl) sulfosuccinate (AOT), and (R,S)-1,2-
O-isopropylidene glycerol (solketal) were supplied by Fluka
(Basel, Switzerland). CaLB had a specific activity of 9.2 units/
mg of protein (1 unit corresponds to the amount of enzyme
that liberates 1 μmol of butyric acid/min at pH 8.0 and 40 °C
using tributyrin as the substrate). (Hydroxypropyl)methyl
cellulose (HPMC) (viscosity 2600−5600 cP, 2% in H2O, 20
°C) and stearic acid (≥99%) were purchased from Sigma
(Germany). All other materials were at least reagent-grade.
Millipore Milli-Q water was used for the preparation of gels and
buffer solution.
2.2. Preparation of AOT/Isooctane Microemulsions
and HPMC MBGs. For the preparation of microemulsions, the
Special Issue: Continuous Processes 14
Received: April 23, 2014
Published: June 27, 2014
© 2014 American Chemical Society
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appropriate amount of CaLB was dissolved in 0.2 M Tris/HCl
buffer (pH 7.5) to a final concentration of 30 mg/mL, and 970
μL of a 0.2 M AOT/isooctane solution and 30 μL of enzyme
solution were added in 2 mL vials. The system was carefully
homogenized until a clear solution was formed. The water
content expressed in terms of the water to surfactant molar
ratio (wo) was 8. The MBGs were prepared by introducing the
appropriate amount of AOT microemulsion containing the
lipase to a second solution of HPMC in water.18 In a typical
experiment, 2 mL of AOT microemulsion containing 1.2 mg of
lipase was gelled with 2.0 g of HPMC and 4.0 mL of water at
room temperature. The esterification reactions took place
under batch and continuous-flow conditions.
2.2.1. Batch Reactions. For either batch or flow experi-
ments, stock solutions of the appropriate fatty acid source
(stearic acid, fatty acid residue, or fatty sewer) with solketal
(100 mM) in the appropriate solvent (heptane, isooctane, or
toluene) were prepared. A 10 mL aliquot of each solution was
added to a 20 mL glass flask, followed by the addition of 7 g of
MBG (containing 1.2 mg of lipase). The system was incubated
at 40−60 °C without stirring. Conversions were analyzed as
described in section 2.3.
Figure 1. Reaction profile for the esterification reaction between
solketal and stearic acid catalyzed by the MBGCaLB system. Conditions:
1:1 stearic acid/solketal (100 mM), 40 °C, no stirring.
show that the immobilized biocatalyst presented excellent
yields of the desired product after only 30 min of reaction,
where the conversion reached a plateau. It is also important to
note that the final conversion yield after 30 min was 90%. This
yield is higher than the one achieved under the same reaction
conditions by the related batch reaction in AOT microemulsion
without the gelling agent.3
Water-in-oil microemulsions present some restrictions
regarding the organic solvents used as far as enhanced solubility
of reaction substrates is concerned. On the contrary, MBGs
operate as heterogeneous catalysts making it possible to apply
an adequate external solvent carrying the substrates for the
desired transformation. In order to evaluate the influence of
nonpolar solvents on the reaction mediated by MBGCaLB, we
performed the same reaction presented in Figure 1 using
different solvents, and the results are shown in Figure 2.
2.2.2. Continuous-Flow Reactions. The reaction mixture
(fatty acid and solketal in the appropriate solvent) was initially
stirred for 5 min at the appropriate reaction temperature.
Esterification reactions were performed in a PBR (Asia Flow
Reactor) equipped with an Omnifit column (cross-sectional
area of 1.7671 cm2 and height of 5 cm = reaction volume of
8.83 mL) containing 7 g of the MBGCaLB (1.2 mg of lipase).
Reaction parameters (temperatures between 40 and 60 °C;
flow rates of 0.1−3.0 mL/min ) were selected on the flow
reactor controller. Initially, only pure solvents were pumped
through, until the instrument was equilibrated at the desired
reaction parameters and stable processing was ensured, after
which the reaction mixture was pumped through the system.
2.3. Conversion Analysis. Process conversions were
measured by determining the residual fatty acid by applying a
modified Lowry and Tinsley method19 and confirmed by GC
analysis. More specifically, 0.3 mL of reaction medium was
placed in vials with 1 mL of reaction solvent and 0.3 mL of a
5% solution of copper acetate/pyridine (pH 6−6.2). The
mixture was vigorously stirred for 30 s. The supernatant was
measured spectrophotometrically at 715 nm. Each reaction was
measured in triplicate, and conversions were calculated by using
calibration curves of the acid used.
Figure 2. Evaluation of solvent effect on the reaction catalyzed by
MBGCaLB. Conditions: 1:1 stearic acid/solketal (100 mM), 40 °C, no
stirring.
For gas chromatography (GC), an HP Innowax column (30
m × 0.25 mm i.d. × 0.32 μm film thickness) mounted on a
Hewlett-Packard (HP) model GC-6890C gas chromatograph
was used. The injector and detector temperatures were 270 °C,
and the oven temperature was constant at 120 °C for 1 min,
then increased by 15 °C/min to 225 °C, and further increased
by 5 °C/min to 260 °C, where it was held constant for 2 min.
As observed in Figure 2, higher conversions were found for
reactions performed in n-heptane. The same result was found
for commercial immobilized enzymes, as described in our
previous publications.20−23 It is obvious that these results can
be related to the solvents’ log P values and their respective
viscosities, which reflect their lipophilicity and can influence the
enzyme stability and the contact surface between the enzyme
and the substrate. Initial rate values were also calculated for the
different solvents used, and the values are shown in Table 1.
The effect of the amount of enzyme loaded in the MBGCaLB
biocatalyst on the reaction rate was also a subject of analysis. As
shown in Figure 3, the initial rate of the reaction increased as
the amount of lipase in the biocatalyst was increased. The
linearity observed under the studied conditions is consistent
with a kinetically controlled enzymatic reaction, maintaining
3. RESULTS AND DISCUSSION
3.1. Batch Reactions. According to our previous work,3
where AOT microemulsions containing CaLB [40 °C, 1:1
stearic acid/solketal (100 mM)] were used, maximum
conversion yields were found after only 30 min of reaction
(80% conversion of protected monoacylglycerol). We used
these conditions in a first attempt to evaluate the performance
of microemulsion-based organogels containing CaLB
(MBGCaLB), and the conversions were analyzed for up to 120
min of reaction (Figure 1). The results presented in Figure 1
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temperatures in a flow reactor filled with MBGCaLB using n-
heptane as the reaction solvent containing stearic acid and
solketal (1:1/100 mM), and the results are presented in Table
2. As can be seen, temperature plays a very important role in
Table 1. Initial rates of the esterification reaction of solketal
and stearic acid catalyzed by MBGCaLB in different solvents
a
solvent
initial rate (mM·min−1
)
isooctane
n-heptane
n-hexane
20.2
27.3
12.4
8.4
Table 2. Effect of residence time (t) on the conversion yields
of the esterification reaction between solketal and stearic
acid (1:1/100 mM) catalyzed by MBGCaLB under
continuous-flow conditions
cyclohexane
a
Reaction conditions: 1:1 stearic acid/solketal (100 mM), 40 °C, no
stirring.
conversion (%)
T (°C)
t = 5.5 min
11 min
55 min
110 min
25
35
40
45
50
8
38
59
69
50
12
42
58
93
54
21
56
25
55
86
77
64
81
>99
66
the esterification reaction catalyzed by MBGCaLB under
continuous-flow conditions. Conversions of 93 and >99%
were found at residence times of 11 and 55 min, respectively, at
45 °C. This result is impressive since in only 11 min a
productivity of 334 g·h−1·(g of enzyme)−1 was revealed, a much
higher value (3.3-fold increase) than that found for the same
reaction under batch conditions. At higher temperatures, a
decrease in conversion yield was observed, as well as greater
turbidity of the samples.
HPMC MBGs are stable up to 65 °C, and at that
temperature the catalytic ability of the enzyme is well-
preserved. However, at temperatures higher than 50 °C a
decrease in the reaction rate can be observed.26 This could be
attributed to structural changes of the gel matrix caused by the
temperature increase that may induce leakage of protein into
the reaction medium.26,27 Allied to the thermal effect, high flow
rates can accelerate this process, resulting in leaching of the
enzyme into the system.
Figure 3. Effect of different concentrations of CaLB, calculated in
terms of milligrams of commercial free lipase in the immobilized
biocatalyst (MBGCaLB), on the rate of the esterification of solketal (100
mM) with stearic acid (100 mM) in n-heptane. Conditions: 40 °C, no
stirring.
the characteristics found for free microemulsion systems.24 The
linearity observed here has been reported before for
Rhizomucor miehei lipase immobilized in similar AOT−
HPMC MBGs for the catalysis of propyl laurate synthesis.18
It is important to emphasize here that both the amount of
enzyme applied in this system and the reaction time required to
reach equilibrium were much lower than for the reaction
performed with the corresponding commercial immobilized
lipase, Novozyme 435 (30 mg pt/g of support).25 This fact can
be more obvious in terms of productivity. Novozyme 435
showed a production of 6.5 (g of ester)·h−1·(g of enzyme)−1,
while the MBGCaLB system gave a value of 101.2 (g of ester)·
h−1·(g of enzyme)−1, confirming the improvement in lipase
performance.
In order to further improve the productivity generated by the
reaction system as described in Table 2, increasing substrate
concentrations were tested in n-heptane while maintaining the
molar ratio of 1:1 (Table 3). The reaction system showed high
Table 3. Effect of increasing substrate concentrations in the
monostearin synthesis catalyzed by the MBGCaLB system
under continuous-flow conditions at 0.5 mL/min (11 min
residence time) and 45 °C
substrate concentration
(mM)
conversion
(%)
productivity g·h−1·(g of enzyme)−1
3.2. Continuous-Flow Reactions. 3.2.1. Reaction of
Solketal with Stearic Acid. Aiming to further improve the
productivity of the MBGCaLB system, we also studied the use of
MBGCaLB under a continuous-flow regime (Scheme 1). Flow
rates between 0.05 and 1.0 mL/min were tested at different
100
150
300
500
750
92
91
91
91
54
241
361
723
1205
225
Scheme 1. Monoacylglycerol synthesis under continuous-
flow conditions with stearic acid and solketal (1:1/100 mM)
in n-heptane and 1.2 mg of lipase
conversions for increasing substrate concentrations up to 500
mM. For substrate concentrations of 500 mM, the final
production was 1205 g·h−1·(g of enzyme)−1, a value that was
the highest for the production of MAG. However, the
recyclability of this system was limited to five cycles (data
not shown), while for lower substrate concentrations (150
mM) the recyclability can reach 15 cycles, as shown in Figure 4.
According to Figure 4, the MBGCaLB system could be reused
for approximately 15 recycles without significant loss of enzyme
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3.2.2. Reactions with Other Fatty Acid Sources. The
optimum conditions as determined by the study presented here
for the reaction of stearic acid and solketal (45 °C, flow rate of
0.1 mL/min) were also applied for the esterification reactions
of palm waste and a sample of fatty sewer as alternative fatty
acid sources. The results are summarized in Table 4. As can be
Table 4. Conversion yields (%) for the esterification
reactions of solketal and two different acid waste sources
catalyzed by MBGCaLB under continuous-flow conditions
with 1:1 acid and solketal in n-heptane and 1.2 mg of CaLB
fatty acid source
substrate concentration (mM)
palm waste
fatty sewer
100
150
300
500
750
83
83
80
51
37
80
75
63
45
29
Figure 4. Recycling of the MBGCaLB system under continuous-flow
conditions.
observed, high conversion yields could be obtained with palm
waste as a fatty acid source, up to a concentration of 300 mM.
The behavior of palm waste is not similar to our previous
results, mainly because of the complexity of this residue, which
contains several impurities that could decrease enzyme
performance. The developed system seemed to be more
affected by the fatty sewer sample since only at a concentration
of 100 mM were good productivities achieved. Although the
chemical compositions of the two acid sources are similar, the
fatty sewer, being a raw material, probably presents some
impurities that affect enzyme performance.
performance. Upon further use of the catalyst (16th to 25th
cycle), a decrease of the conversion yield was noticed as a result
of the large amount of protein in the collected samples, as
measured by the Bradford method.28 The ability to reuse the
catalyst 15 times is an important advantage of the MBGCaLB
system applied under continuous-flow conditions.
Taking into account the fact that the productivity is an
important parameter, we compared the values obtained in this
study for batch and continuous-flow reactions with the ones
obtained using other commercial immobilized enzymes. The
results (summarized in Figure 5) show that MBGCaLB can be
much more efficient than the most traditional immobilized
enzymes used in the literature for esterification reactions under
batch conditions. Under continuous-flow conditions, the results
obtained for Novozyme 435 and MBGCaLB are similar.
Nevertheless, MBGCaLB shows many advantages over commer-
cial immobilized enzymes, such as the versatility of the amount
of enzyme loaded on the biocatalyst, biocompatibility of the
support, and reusability, among others.
4. CONCLUSION
In the work presented here, we have shown that CaLB lipase
immobilized in an MBG matrix can efficiently be used to
catalyze the production of protected MAG, since high
productivities were obtained compared with commercial
immobilized lipases. Under continuous-flow conditions, all of
the reagents could be converted to protected MAG in a
Figure 5. Relation between productivity and catalysis system for the esterification of stearic acid and solketal at 100 mM in n-heptane.
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residence time of 11 min at 45 °C, giving conversion yields of
up to 99%. The study of the reuse of the system showed that it
can be recycled 15 times without activity loss, a number 2 times
higher than that for immobilized lipase under batch
a
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AUTHOR INFORMATION
Corresponding Author
*E-mail: souzarod21@gmail.com.
■
Notes
The authors declare no competing financial interest.
ADDITIONAL NOTE
Although there is a relatively small difference between the
■
a
structures of the applied organic solvents, these differences
impart intense chemical properties, such as viscosity and
lipophilicity. More viscous solvents tend to reduce the surface
contact between the biocatalyst and the substrate, resulting in
lower conversions. However, the enzyme affinity for the solvent
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interface formation to expose its active site and perform
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