Evaluation of diatomaceous earth supported lipase sol-gels as a medium for enzymatic transesterification of biodiesel

Article abstract of DOI:10.1016/j.molcatb.2012.01.014

Immobilized lipase has the potential to be the catalyst of choice for biodiesel production since it is efficient, effective, and environmentally friendly; however, the stability and activity of lipase must be addressed before enzymatic biodiesel production processes can be industrially accepted. This study investigates an enzyme immobilization procedure that immobilizes lipase in a sol-gel supported on diatomaceous earth (Celite R632), and determines its potential for biodiesel production in terms of achievable conversion and apparent stability. Four immobilized materials (lipase sol-gels with and without Celite at two protein loading levels) were compared in terms of their immobilized protein content, conversion of methanol to methyl oleate, lipase activity, long term stability, and glycerol-water adsorption. The Celite R632 sol-gel with high protein loading achieved the maximum conversion in the 6-h reaction period (90%). A drying step was found to be advantageous prior to the reaction, and the absorption of glycerol-water on the Celite was only found to be significant at high levels of glycerol. The material was found to be very stable upon storage at 4 °C for up to 1.5 years, losing only about 15% of its percent conversion capacity per year. Based on this study, the supported immobilization technique shows significant potential as a novel catalyst for biodiesel production.

Full text of DOI:10.1016/j.molcatb.2012.01.014

Journal of Molecular Catalysis B: Enzymatic 77 (2012) 92–97
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Evaluation of diatomaceous earth supported lipase sol–gels as a medium for
enzymatic transesterification of biodiesel
S.M. Meunier∗, R.L. Legge
Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1
a r t i c l e i n f o
a b s t r a c t
Article history:
Immobilized lipase has the potential to be the catalyst of choice for biodiesel production since it is efficient,
effective, and environmentally friendly; however, the stability and activity of lipase must be addressed
before enzymatic biodiesel production processes can be industrially accepted. This study investigates
an enzyme immobilization procedure that immobilizes lipase in a sol–gel supported on diatomaceous
earth (Celite® R632), and determines its potential for biodiesel production in terms of achievable con-
version and apparent stability. Four immobilized materials (lipase sol–gels with and without Celite® at
two protein loading levels) were compared in terms of their immobilized protein content, conversion
of methanol to methyl oleate, lipase activity, long term stability, and glycerol–water adsorption. The
Celite® R632 sol–gel with high protein loading achieved the maximum conversion in the 6-h reaction
period (90%). A drying step was found to be advantageous prior to the reaction, and the absorption of
glycerol–water on the Celite® was only found to be significant at high levels of glycerol. The material was
found to be very stable upon storage at 4 ◦C for up to 1.5 years, losing only about 15% of its percent con-
version capacity per year. Based on this study, the supported immobilization technique shows significant
potential as a novel catalyst for biodiesel production.
Received 26 October 2011
Received in revised form 12 January 2012
Accepted 15 January 2012
Available online 25 January 2012
Keywords:
Enzyme immobilization
Biodiesel
Sol–gel
Transesterification
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
sol–gel immobilization [3–5]. Further, ␻-transaminases encap-
sulated via a supported immobilization procedure with Celite®
Lipase is a triacylglycerol hydrolase and is responsible for the
hydrolysis of ester bonds in triglycerides. At hydrophobic inter-
faces, lipases are known to undergo interfacial activation which
causes a surge in enzymatic activity [1]. Enzymatic reactions typ-
ically have many advantages such as enhanced specificity and
efficiency, however, decreased activity and stability are common
elements of concern when working with enzymes.
One way to extend the operational stability, and thus decrease
the effective cost of the enzyme, is by immobilization. Although
biodiesel production with alkali catalysts is known to be the least
costly option, processes using immobilized lipase are significantly
less costly than those using free lipase [2]. Jegannathan et al. also
show that if immobilized lipase is reused more than five times or
the production cost of the lipase is reduced significantly, enzymatic
biodiesel production could be competitive with the alkali biodiesel
process [2].
sol–gels exhibit enhanced activity even at extreme conditions such
as high pH and temperature, and can be recycled up to eight times
without dramatically reducing the achievable conversion [6]. Other
studies comparing Celite® sol–gels show similar results using lipase
as the enzyme of interest [7,8].
Due to environmental and economical concerns, biodiesel has
become an invaluable alternative to standard diesel fuels [9].
Biodiesel is comprised of fatty acid alkyl esters and is produced
via the transesterification of oils. Conventionally, biodiesel is pro-
duced using either an alkali or acid catalyst, but using lipase as the
catalyst for transesterification could provide significant advantages
over these traditional processes. Enzymatic processes self-adapt to
changes in raw material quality, produce biodiesel in few process-
ing steps, use little energy, produce little wastewater, and yield
high quality by-products [10]. The challenges associated with enzy-
matic transesterification include low reaction rates, high enzyme
cost, and the potential for enzyme deactivation [11].
Since methanol, glycerol and water are all known to have
inhibitive effects on lipase each of these components must be
closely monitored in the biodiesel production process. Methanol is
a substrate as well as a lipase inhibitor in the biodiesel production
process; therefore, lower than stoichiometric ratios of methanol are
commonly used for biodiesel production to avoid enzyme deac-
tivation [12–16]. Glycerol is a by-product of enzymatic biodiesel
Enzyme entrapment in polymers such as sol–gels provides a
stable enzyme support with very strong bonds while not requir-
ing complex chemical preparation [3]. Both the thermal and
chemical stability and activity of enzymes can be improved via
∗
Corresponding author. Tel.: +1 519 888 4567x31648; fax: +1 519 888 4347.
E-mail address: sarah.meunier@uwaterloo.ca (S.M. Meunier).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2012.01.014

S.M. Meunier, R.L. Legge / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 92–97
93
Table 1
solution. For the high lipase concentration Celite sol–gels, the lipase
solution was used undiluted at a concentration of approximately
12 mg/mL. The resultant mixture was added to the support mate-
rial with approximately 3 mL sol–gel mixture for every 2 g of Celite®
R632. After thorough mixing, the sol–gel Celite was deposited in
a Petri dish, sealed and aged at 4 ◦C for 24 h. The Celite® sol–gel
was then dried uncovered at 4 ◦C until the drying rate was less
than 1 mg/h. Finally, the supported gel was removed from the
Petri dish and washed twice with phosphate buffer (50 mM, pH
7.0, 5 mL buffer per gram of sol–gel for each wash) to remove
any protein that was not completely immobilized within the gel.
Excess solvent was evaporated from the gel at room tempera-
ture overnight prior to storing the gels in a sealed container at
4 ◦C.
Unsupported gels were prepared in a similar manner except
that the evaporated precursor and enzyme mixture was deposited
directly into the Petri dish for aging and drying, the dried sol–gel
was crushed in a mortar upon removal from the Petri dish, and the
washing solutions were separated from the sol–gel by centrifuga-
tion.
Description of the lipase preparations used for analysis in terms of the support
material, sol–gel formulation, and lipase solution concentration.
Lipase
preparation
Support
material
Sol–gel
Lipase
solution
C-SG-4
C-SG-12
U-SG-4
U-SG-12
Free
Celite® R632
Celite® R632
Unsupported
Unsupported
Unsupported
80% PTMS/20% TMOS
80% PTMS 20% TMOS
80% PTMS/20% TMOS
80% PTMS/20% TMOS
No sol–gel
4 mg/mL
12 mg/mL
4 mg/mL
12 mg/mL
4 mg/mL
production that is inhibitory to the lipase, but glycerol produced
during transesterification can be absorbed by silica beds [17–19].
In terms of water absorption, according the Celite® supplier, World
Minerals, the water absorption capacity of Celite® is up to 500%
by weight, depending on the type of Celite® considered. This is a
crucial parameter since, although water is necessary for lipase acti-
vation in biodiesel production, excess water levels will inhibit the
lipase by occupying the enzyme support pore space and limiting
contact between the enzyme and substrates [9].
This study considers the enzymatic activity of lipase immobi-
lized in Celite® R632 sol–gels based on a supported immobilization
scheme. A comparison is made between the achievable enzymatic
conversion of methanol to methyl oleate for unsupported sol–gels
and sol–gels supported on Celite® R632 at both high and low
protein content levels. Further, the glycerol–water absorption is
considered for both Celite® R632 and sol–gel Celite® R632 using
thermogravimetric analysis in an attempt to elucidate the possi-
ble effects of glycerol and water in a biodiesel process for lipase
immobilized in Celite® R632 sol–gels. This information is valuable
in evaluating the potential of Celite® R632 sol–gels as a supported
immobilization medium for enzymatic biodiesel production.
2.2.2. Protein measurement
The total protein content of the gels was calculated using a mass
balance of the protein content of the enzyme solution loaded to the
sol–gels and content in the two buffer wash solutions. The degree of
immobilization was calculated as the percentage of protein in the
sol–gel compared to the amount of protein desired in the supported
sol–gel. The amount of protein was quantified using a Varian HPLC
system (Varian Inc., Mississauga, ON) equipped with an Agilent Zor-
bax Bio Series GF-250 column (Agilent Technologies, Mississauga,
ON) and calibrated using a BCA protein assay kit (Pierce Biotechnol-
ogy Inc., Rockford, IL). The mobile phase for the HPLC analysis was
200 mM phosphate buffer (pH 7.0) and detection at a wavelength
of 280 nm.
2. Experimental
2.1. Materials
2.2.3. Enzymatic lipid transesterification
The enzymatic activity of the supported lipase sol–gels was
determined by GC–MS analysis. The reactions were carried out at
40 ◦C with agitation for 6 h. The reaction mixture consisted initially
of approximately 1 g of the supported lipase sol–gel, 4 mmol of
triolein and 4 mmol of methanol (total reaction volume 3.89 mL).
Each hour, a 10 ␮L sample was removed from the reaction vial
and diluted in 990 ␮L hexane with 100 ␮L of the internal standard,
HDA-ME. The formation of methyl oleate, the reaction product,
was followed using a Varian GC–MS system (CP-3800 gas chro-
matograph, Saturn 2000 mass spectrometer/mass spectrometer)
equipped with a CP-Wax 52 CB fused silica column (CP8513, Varian
Inc., Mississauga, ON). 1 ␮L samples of the diluted reaction mixture
were injected into the GC at an injector temperature of 250 ◦C and
a split ratio of 50. Helium was used as the carrier gas with a col-
umn flow of 1 mL/min. The GC oven temperature was initially set to
170 ◦C for 10 min, ramped at 10 ◦C/min to 250 ◦C, and held at 250 ◦C
for 2 min.
Celite® samples were a gift from World Minerals (Santa Barbara,
CA). Lipase (NS44035) was a gift from Novozymes North America
Inc. (Franklinton, NC). The biological source of NS44035 cannot be
disclosed by the supplier; the activity of NS44035 is 20,000 PLU/g.
Tetramethyl orthosilicate (TMOS), trimethoxypropylsilane (PTMS),
triolein, glycerol, methyl oleate and methyl heptadecanoate (HDA-
ME) were obtained from Sigma–Aldrich Canada Ltd. (Oakville, ON).
Acetonitrile was obtained from EMD Chemicals (Gibbstown, NJ).
Sodium phosphate was obtained from Mallinckrodt Baker (Phillips-
burg, NJ). Hexane and hydrochloric acid were obtained from Fisher
Scientific Company (Ottawa, ON). Ultrapure water was produced
using a Milli-Q water purification system from Millipore (Billerica,
MA). All other chemicals were obtained from local suppliers.
2.2. Methods
2.2.1. Immobilization of lipase
The enzymatic activity of the dried sol–gel formulations were
carried out in the same manner as the original gels with the excep-
tion that prior to the enzymatic assay the gels were dried overnight
in a 60 ◦C oven. At this point, no further change in mass was
observed from the drying process and thus any remaining water
was assumed to be completely removed from the sol–gel formula-
tion.
Using the GC–MS method described, a calibration was com-
pleted based on a methyl oleate standard (40–800 mM) and used as
the basis for all methyl oleate concentrations provided. From the
GC–MS chromatograms, methyl oleate was the only visible peak
indicating that no side reactions occurred. The enzymatic activity
of the lipase was determined from the slope of the methyl oleate
Four different lipase sol–gels were produced for analysis—with
and without Celite® R632 as a support material and with high and
low concentrations of lipase (Table 1).
To immobilize lipase in diatomaceous earth sol–gels, 0.08 mol
PTMS and 0.02 mol TMOS were hydrolyzed in the presence of
0.1 mol ultrapure water and 200 ␮L HCl (0.1 M). The mixture was
sonicated for 1 h to allow for complete hydrolization of the pre-
cursors. The precursor solution was then rotary evaporated in a
heated water bath at 40 ◦C for 30 min to remove excess water
and alcohol. A solution of lipase and phosphate buffer (50 mM,
pH 7.0) with an approximate protein concentration of 4 mg/mL
was prepared, and 14 mL was added to the hydrolyzed precursor

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S.M. Meunier, R.L. Legge / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 92–97
Fig. 1. Protein content and degree of protein immobilization for four sol–gel for-
mulations. Error bars represent a 95% confidence interval.
Fig. 2. Percent conversion of methanol to methyl oleate based on a 6-h batch
reaction for each sol–gel formulation without and with a drying step prior to the
enzymatic assay. Error bars represent a 95% confidence interval.
concentration–time profile over the weight of immobilized enzyme
material used.
decreases slightly for both the Celite® and unsupported sol–gels.
Since there is excess lipase in triple lipase sol–gels, there is more
opportunity for lipase that is incompletely entrapped to be easily
removed during the washing steps.
The high protein content unsupported sol–gel (U-SG-12) did
exhibit much more variability than the other immobilization
regimes (Fig. 1). This is likely caused by the potential for aggre-
gation, incomplete immobilization and lipase deactivation during
the sol–gel formation procedure. With such high levels of protein
and without the Celite® support material, the lipase is much more
exposed and thus more sensitive to environmental changes. This
enhanced potential for enzyme deactivation and poor immobiliza-
tion can conceivably greatly reduce the reproducibility of the lipase
sol–gel immobilization procedure.
2.2.4. Desorption of glycerol and water
The desorption of glycerol and water from Celite® R632 upon
equilibration was measured using a thermogravimetric analyzer
(Q500 TGA, TA Instruments, New Castle, DE). Prior to analysis, each
sample was immersed in the desired solution containing glycerol
and water at various concentrations (0%, 10%, 25%, 50%, 75% and
100% by volume glycerol) and equilibrated for 24 h. The sample was
then washed with water to remove excess glycerol and air dried
overnight. The TGA analysis method ramped the temperature from
30 ◦C to 400 ◦C at 10 ◦C/min followed by air cooling for 10 min under
50 mL/min N2 gas. The onset of desorption (temperature at which
0.025% mass loss is achieved), the peak desorption rate (maximum
mass loss rate achieved), the peak temperature (temperature at the
peak desorption rate), and the total mass loss (percent mass lost at
the end of the analysis) were determined from the weight loss curve
and the derivative weight loss curve.
3.2. Enzymatic properties
Free lipase at a comparable level to that found in the immobi-
lized lipase preparation C-SG-4 was assayed to determine a base
methyl oleate conversion level for comparison to the immobilized
preparations. Based on GC–MS analysis, no measurable methyl
oleate was detected during the 6 h reaction.
3. Results and discussion
3.1. Physical properties
Based on the percent conversion after 6 h for methyl oleate
production (Fig. 2), the Celite® R632 sol–gels exhibited an
increased conversion when the amount of lipase loaded onto
the gels was tripled. In addition, the Celite® supported sol–gels
achieved slightly more conversion when dried due to an
excess of water absorbed on the support material since Celite®
is absorbent and lipase is inhibited by excess water con-
tent.
Comparing the protein content and the degree of immobiliza-
tion of the four different lipase sol–gels formed, several phenomena
were observed (Fig. 1). First, for both the low and high protein level
sol–gels, the unsupported sol–gels have higher protein contents in
comparison to the Celite® supported sol–gels. Since there is addi-
tional material added to the Celite® sol–gels, lower protein content
per gram is expected for these gels.
The protein level immobilized in the sol–gel was not propor-
tional to target load for the sol–gel during the formation procedure
(Fig. 1). For the Celite® sol–gels the protein content was approx-
imately double when triple the lipase was loaded, but for the
unsupported sol–gels the protein content was approximately triple
as expected. One possible explanation for the lack of proportional-
ity for the Celite® sol–gels is that the excess of lipase in the triple
unsupported formulation caused protein aggregation and subse-
quent immobilization. This effect might not be as pronounced for
the lower levels of lipase because there is not enough lipase in the
formulation and for the triple lipase Celite® system because the
presence of Celite® reduces the apparent protein concentration.
The observed degree of protein immobilization for the Celite®
sol–gels is comparable to that for the unsupported sol–gels (Fig. 1).
When the amount of lipase is tripled, the degree of immobilization
The unsupported sol–gels demonstrate the opposite trend—the
sol–gel preparation with triple lipase achieves a lower percent
conversion than the regular lipase loading and the dried gel prepa-
rations have lower conversion than those that are not dried. As
discussed with regards to the protein content of the gels, this
formulation does have much more variability than the other for-
mulations which could be caused by aggregation, incomplete
immobilization, and deactivation of the protein due to the excess
quantities of protein. Since there is no Celite® present in the
unsupported sol–gels to remove the water, drying the enzyme
preparation may result in inactivation of the lipase rather than
removing the excess water and thereby causing the reduction in
product concentration.
In comparison to the Celite® supported sol–gel, adding neat
Celite® to the unsupported sol–gel (SG + Celite®) greatly reduces

S.M. Meunier, R.L. Legge / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 92–97
95
120%
100%
80%
60%
40%
20%
0%
0
100
200
300
400
500
Time (days)
Fig. 3. The enzymatic activity on a per gram of material basis for each sol–gel formu-
lation as determined from the conversion of methyl oleate and the protein content
of the gels. Both the sol–gel formulations without and with a drying step prior to
the reaction were considered. Errors bars represent a 95% confidence interval.
C-SG-4
U-SG-4
Linear (U-SG-4)
Linear (C-SG-4)
Fig. 4. Average percent conversion of methanol to methyl oleate for sol–gels after
storage at 4 ◦C for unsupported (ꢀ) and Celite® R632 supported sol–gels (ꢁ). The
lines represent the lines of best fit for the unsupported sol–gel (broken) and the
Celite® R632 supported sol–gel (solid). Error bars represent a 95% confidence inter-
val.
the percent conversion likely due to reactant, product and water
absorption by the Celite® thereby preventing the reaction from
progressing. Comparing the supported and unsupported sol–gel
formulations, the addition of the Celite® as a support material for
the sol–gel increases the percent conversion to methyl oleate for
both sol–gel formulations.
Comparing the activity of the different sol–gel preparations on
a mass basis (Fig. 3) reveals that the dried unsupported and sup-
ported sol–gels all have comparable activities. This demonstrates
the beneficial effects of using Celite® and providing a support since
the Celite® sol–gels contain much less enzyme per gram of material
in comparison to the unsupported sol–gels (Fig. 1). The unsup-
ported sol–gel preparation with the higher level of lipase (U-SG-12)
does have a slightly lower activity in comparison with the other
materials when dried which is likely due to the excess lipase in
the preparation which is either inactive and/or inaccessible to the
substrates.
The unsupported sol–gels have a much higher activity than the
Celite® sol–gels when assayed without a drying step. The adsorp-
tive properties of the Celite® and the inhibition of lipase caused by
excess water are the expected causes of this phenomenon. Despite
the increased lipase content (Fig. 1) in the high concentration lipase
unsupported sol–gel (U-SG-12), the activity is comparable to the
regular lipase level unsupported sol–gel (U-SG-4). Therefore, the
excess lipase in this system may not be accessible to the substrates
or has been rendered inactive.
onset of desorption, peak desorption rate, peak temperature, and
total mass loss.
Based on the weight change with respect to temperature data
obtained from the TGA analysis, the temperature at which desorp-
tion was established for each case (10%, 25%, 50%, 75%, and 100%
glycerol for both neat Celite® and the Celite® sol–gel C-SG-4, data
not shown) was compared. The average onset of desorption for all
cases was 113.3 1.2 ◦C. This indicates that the observed desorp-
tion is caused by a combination of the water and the glycerol rather
than the components separately.
The peak desorption rate obtained from the TGA analysis is a
measure of the level of adhesion of solvent to the Celite® or Celite®
sol–gel with respect to the cohesion of solvent molecules to each
other. High peak desorption rates indicate that the solvent coheres
more strongly to itself and low peak desorption rates signify that
the solvent molecules adhere very well to the support material.
The total mass loss indicates the absorptive capacity of the Celite®
or Celite® sol–gel with respect to the applicable glycerol–water
Peak Desorpꢀon
100
98
96
94
92
90
88
86
84
82
80
0.4
0.35
0.3
The Celite® supported sol–gels and the unsupported sol–gels
were also compared based on their stability over a period of approx-
imately 1.5 years (Fig. 4). The unsupported and Celite® supported
sol–gel formulations show almost identical trends with a very grad-
ual decrease in product output over time. The unsupported sol–gel
(U-SG-4) has a 0.05% decrease in product concentration per day
while the Celite® supported sol–gel (C-SG-4) has a 0.04% decrease
per day. This is about an 18% (U-SG-4) and 15% (C-SG-4) decrease
in product concentration per year. This indicates that a very stable
enzyme formulation has been developed with a long shelf life when
stored at 4 ◦C.
Total Mass Loss
0.25
0.2
0.15
0.1
0.05
0
400
0
50
100
150
200
250
300
350
Temperature (oC)
3.3. Absorptive properties
Weight
Derivaꢀve Weight
A TGA spectrum (Fig. 5) shows typical weight-time and deriva-
tive weight-time profiles for a sample of Celite® R632 sol–gel
(C-SG-4). The peak desorption and total mass loss parameters are
indicated on the graph. These profiles are used to determine the
Fig. 5. Typical TGA profile including the sample weight (solid) and the derivative
weight (broken). The peak desorption point and the total mass lost are indicated
on the graph. The sample shown is Celite® R632 sol–gel (C-SG-4) at 50% glycerol
equilibrating solution.

96
S.M. Meunier, R.L. Legge / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 92–97
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
200
190
180
170
160
150
140
130
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Glycerol / Water Equilibraꢀng Soluꢀon (v% glycerol)
Glycerol / Water Equilibraꢀng Soluꢀon (v% glycerol)
C-SG-4
Neat Celite
C-SG-4
Neat Celite
Fig. 6. Peak desorption rates for Celite® R632 sol–gels (ꢀ) and plain Celite® R632
(ꢁ) based on different levels of glycerol in the equilibrating solution. Error bars
represent a 95% confidence interval.
Fig. 8. Peak desorption temperatures for Celite® R632 (ꢀ) sol–gels and plain Celite®
R632 (ꢁ) based on different levels of glycerol in the equilibrating solution. Error bars
represent a 95% confidence interval.
The peak desorption temperature as determined by the TGA is
a measure of the strength of cohesion of the glycerol–water solu-
tion to the Celite® or Celite® sol–gel material. The peak desorption
temperatures for each solution were different (Fig. 8), ranging from
132 ◦C to 187 ◦C, and showed similar trends to those observed for
the peak desorption rate (Fig. 6) and the total mass loss (Fig. 7).
One phenomena observed is the similarity between the values for
the 100% glycerol solution absorption when comparing the reg-
ular Celite® and the sol–gel supported Celite® (Fig. 8), indicating
that the difference caused by the sol–gel is most prominent when
water is present in the equilibrating solution. According to Clif-
ford and Legge, both contact angle measurements and TGA indicate
that PTMS–TMOS sol–gels are hydrophobic and that increasing the
proportion of PTMS increases the hydrophobicity of the sol–gels
[20]. Therefore, the hydrophobicity of the sol–gel appears to have
a stronger effect when pure water is used as the equilibrating solu-
tion rather than when glycerol–water solutions are used.
These phenomena are important in a biodiesel production pro-
cess since glycerol is a by-product of the reaction. The highly
absorptive properties of the Celite® sol–gel will inhibit the lipase
and prevent the reaction from proceeding. Care must be taken to
ensure that glycerol is removed when the reaction is run on a
continuous basis. However, at low levels of glycerol (i.e. 10% by vol-
ume), very little glycerol is absorbed in the Celite® sol–gel material
(approximately 2.5% by mass), so this phenomena becomes more
of a concern as the concentration of glycerol increases.
solution. At the end of the TGA analysis, any glycerol and water
absorbed and retained by the material are subsequently desorbed
and thus quantified by the total mass loss.
Considering both the peak desorption rate (Fig. 6) and the total
mass loss (Fig. 7) when neat Celite® and Celite® sol–gels are incu-
bated in glycerol–water solutions similar trends are evident. Both
the peak desorption rates and the total mass loss are higher in each
case for the neat Celite® than for the Celite® sol–gel. It is likely that
the sol–gel provides a protective barrier that reduces absorption of
the glycerol–water solution.
Additionally, the amount of solution absorbed does generally
increase with increasing percentage of glycerol in the equilibrating
solution. One notable exception is the decrease for the neat Celite®
from 75% glycerol to 100% glycerol. This shows that the water is
necessary for absorption of glycerol onto the Celite. However, this is
not the case for the Celite® sol–gel. The increasing adsorption trend
of the supported sol–gel material is not affected by eliminating the
water from the equilibrating solution.
When the equilibrating solution consisted solely of water, there
was no evidence of desorption from the neat Celite® or sol–gel
Celite® (Figs. 6 and 7). This is evidence that the water is not readily
retained by the Celite® material despite its absorptive properties.
30
25
20
15
10
5
4. Conclusions
A procedure to immobilize lipase in sol–gels supported on
Celite® R632 was developed and shown to be valuable for the
transesterification of triolein to produce methyl oleate. Three main
properties were considered for comparison: protein loading, enzy-
matic activity, and glycerol–water adsorption, for four different
sol–gel preparations: unsupported sol–gel low protein level (U-SG-
4), Celite® sol–gel low protein level (C-SG-4), unsupported sol–gel
high protein level (U-SG-12), and Celite® sol–gel high protein level
(C-SG-12).
Based on the amount of protein retained with respect to that
loaded onto the sol–gel, the immobilization procedure is more
effective (i.e. incomplete protein immobilization is minimized) at
the low protein levels. The water absorbed by the Celite® during
sol–gel preparation must be removed as it inhibits the lipase–the
maximum production of methyl oleate (80% in 6 h) was achieved
0
0
10
20
30
40
50
60
70
80
90
100
Glycerol / Water Equilibraꢀng Soluꢀon (v% glycerol)
C-SG-4
Neat Celite
Fig. 7. Total percentage mass loss for Celite® R632 sol–gels (ꢀ) and plain Celite®
R632 (ꢁ) based on different levels of glycerol in the equilibrating solution. Error
bars represent a 95% confidence interval.

S.M. Meunier, R.L. Legge / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 92–97
97
with the high protein content Celite® sol–gel (C-SG-12) after a pre-
drying step. Over a 1.5-year time frame, both the unsupported and
Celite® supported sol–gels exhibited high stability when stored at
4 ◦C. The sol–gel was found to provide a protective barrier that
inhibits the absorption of the glycerol–water solution. Therefore,
the removal of glycerol is key in preventing the inhibition of the
lipase, but is only a concern at very high levels of glycerol (approx-
imately 75% glycerol by volume).
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Acknowledgements
This work was supported by the Natural Sciences and Engi-
neering Research Council (NSERC) in the form of a Postgraduate
Scholarship to SMM and a Discovery Grant to RLL. We thank
Novozymes North America for supplying samples of the lipase for-
mulation and World Minerals for supplying samples of the Celite®
support materials.
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