Immobilization of Rhizopus oryzae lipase on silica aerogels by adsorption: Comparison with the free enzyme

Article abstract of DOI:10.1016/j.procbio.2011.01.029

Rhizopus oryzae lipase (ROL) was immobilized by physical adsorption onto silica aerogels. The functional properties of immobilized lipase were determined and compared to the soluble lipase ones. The optimum temperature for both free and immobilized lipase activities was 37 °C. We found that the immobilization of R. oryzae lipase onto silica aerogels increased remarkably its stability at high temperatures and within a wade pH range. Besides the immobilized enzyme exhibited a high tolerance to apolar solvent and retained its fully activity in suspension after 4 months of storage at 4 °C. This immobilized biocatalyst is applied in n-butyl oleate synthesis by esterification of oleic acid with n-butanol, using hexane as an organic solvent. The best conversion yield of the ester butyl oleate was obtained with the immobilized lipase (80% versus 35% with the free lipase). This catalytic esterification has been carried on the presence of hexane at 37 °C with oleic acid to butanol molar ratio of 1:1 and 450 IU of immobilized lipase. Furthermore, the reuse of the lipase immobilized by adsorption allowed us to observe that its can achieved 12 successive cycles, without a significant loss of its catalytic activity. Such results revealed good potential for recycling under non-aqueous system.

Full text of DOI:10.1016/j.procbio.2011.01.029

Process Biochemistry 46 (2011) 1083–1089
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Immobilization of Rhizopus oryzae lipase on silica aerogels by adsorption:
Comparison with the free enzyme
Nadia Kharrat , Yassine Ben Ali , Sana Marzouk , Youssef-Talel Gargouri , Maha Karra-Châabounia
a
a
b
a,∗
a
Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS route de Soukra, BPW, 1173 Sfax-Tunisia, Tunisia
Laboratoire de chimie industrielle II, ENIS route de Soukra, BPW, 1173 Sfax-Tunisia, Tunisia
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 September 2010
Received in revised form
Rhizopus oryzae lipase (ROL) was immobilized by physical adsorption onto silica aerogels. The functional
properties of immobilized lipase were determined and compared to the soluble lipase ones. The optimum
◦
temperature for both free and immobilized lipase activities was 37 C. We found that the immobilization
2
9 December 2010
of R. oryzae lipase onto silica aerogels increased remarkably its stability at high temperatures and within a
Accepted 25 January 2011
wade pH range. Besides the immobilized enzyme exhibited a high tolerance to apolar solvent and retained
◦
its fully activity in suspension after 4 months of storage at 4 C. This immobilized biocatalyst is applied in
Keywords:
n-butyl oleate synthesis by esterification of oleic acid with n-butanol, using hexane as an organic solvent.
Rhizopus oryzae lipase
Immobilization
Adsorption
The best conversion yield of the ester butyl oleate was obtained with the immobilized lipase (80% versus
◦
3
5% with the free lipase). This catalytic esterification has been carried on the presence of hexane at 37 C
Silica aerogels
Stability
Esterification
with oleic acid to butanol molar ratio of 1:1 and 450 IU of immobilized lipase. Furthermore, the reuse of
the lipase immobilized by adsorption allowed us to observe that its can achieved 12 successive cycles,
without a significant loss of its catalytic activity. Such results revealed good potential for recycling under
non-aqueous system.
©
2011 Elsevier Ltd. All rights reserved.
1
. Introduction
Silica aerogels can be considered as the suitable carrier for biocat-
alysts immobilization.
Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) are among
The selection of an immobilization strategy is based on effective-
ness of enzyme utilization, cost of the immobilization procedure,
toxicity of immobilization reagents and the desired final proper-
ties of the immobilized biocatalyst [11]. The immobilization could
resolve some problems when using enzymes as industrial bio-
catalysts: enzyme recovery, enzyme stability, enzyme selectivity,
inhibition by the medium or products. In all these cases, the use
of a battery of immobilization procedure permits the control the
support-enzyme interaction and increases the possibilities of suc-
cess. Among immobilization techniques, adsorption may have the
highest commercial potential compared to other techniques due
to its relatively low cost, simpliness and it allows retaining of
high catalytic activity. This method offers the reusability of expen-
sive supports after inactivation of immobilized enzyme [12–14].
However, adsorption is generally not a very tight interaction and
the protein will desorbs from support during washing and other
steps. Thus, immobilization via adsorption requires an electrostatic
interaction between support and enzyme. The lipase immobiliza-
tion by adsorption on various supports was reported in numerous
studies. Macroporous resin was used as support for the immo-
bilization of Candida sp. 99–125 lipase by physical adsorption
the most popular enzymes in biocatalysis with a broad variety of
applications in fine chemistry, pharmaceutical industry and in the
food industry owing to their usefulness in both hydrolytic and
synthetic reactions [1,2]. Most of lipases used in industrial pro-
cesses were in the immobilized state. Indeed, the immobilization of
enzymes to solid support materials is of great interest due to some
important advantages such as: improvement of the activity and sta-
bility of enzymes, possibility recovery of the immobilized enzyme
at the end of the reaction and thus its potential reuse and accom-
plishment of the enzyme-catalyzed reaction under a continuous
process [3]. Numerous papers reported different lipases immobi-
lization techniques such as physical adsorption of the enzyme on
a carrier material [4], its entrapment or microencapsulation in a
solid support [5,6] and covalence binding to a solid matrix [7,8].
Typical materials used for these purposes include chitin, cellu-
lose, polyalanine and silica [9]. Silica aerogels are extremely porous
materials with high specific surface areas. It exhibits interesting
physico-chemical properties, including very high specific surface
2
−1
areas (500–1000 m g ) [10]. Due to economical considerations,
[
15]. Foresti and Ferreira [16] reported the immobilization of Can-
dida antarctica B lipase, by adsorption, onto polypropylene coated
glass balls, and used to synthesize ethyl oleate. Torres et al. [17]
∗ Corresponding author. Tel.: +216 74 675055; fax: +216 74 675055.
E-mail address: ytgargouri@yahoo.fr (Y.-T. Gargouri).
1
359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2011.01.029

1
084
N. Kharrat et al. / Process Biochemistry 46 (2011) 1083–1089
reported the immobilization of the same lipase by adsorption on
polyethylene–agarose.
2.6. Characterization of immobilized lipase
2
.6.1. Effect of temperature on the free and the immobilized lipase activities and
In this study, we describe the physical adsorption of Rhizopus
oryzae lipase (ROL) isolated and produced in our laboratory [19],
onto silica aerogels. The stability and the activity of the immobilized
lipase were investigated. Finally, we have tested the ability of this
enzyme to synthesize 1-butyl oleate used in industrial applications.
stabilities
The effect of temperature on the relative activity of the free and immobilized
ROL was determined at pH 8.5 in temperature range varying from 25 to 50 C, using
olive oil emulsion as substrate. Relative activities were calculated as the ratio of the
enzyme activity measured at different temperatures to the maximal activity of the
enzyme measured as described above.
◦
The thermal stability assays were performed by the incubation for 1 h of the
◦
same amount of immobilized or free ROL at various temperatures (25–60 C). After
2
. Materials and methods
cooling to room temperature, the activity of enzyme was measured under standard
conditions (pH 8.5, 37 C) as described above. Residual activities were calculated as
the ratio of the activity of enzyme measured after incubation to the maximal activity
of the enzyme.
◦
2.1. Materials
The n-hexane, the 1-butanol, and the dioxane (99.5% of purity) were purchased
from Prolabo (Paris, France). The oleic acid was purchased from Fluka (Buchs,
Switzerland). The pH-Stat was from Metrohm (Herisau, Switzerland). The shaker
2
.6.2. Effect of pH on the free and the immobilized lipases activities and stabilities
The effect of pH on the free and immobilized lipase activities was studied in
(
Certomat H/HK) was from B. Braun Biotech (Goettingen, Germany). The spec-
◦
the pH range varying from 7.5 to 11 at 37 C using olive oil emulsion as substrate.
Relative activities were calculated as the ratio of the activity of enzyme measured
at different pH to the maximal activity of enzyme, at pH 8.5 and 37 C.
trophotometer UVmini 1240 was from Shimadzu (Japan). The centrifigeuse was
from Hermle (Germany).
The strain was isolated from olive in decomposition and it was identified as R.
Oryzae [19].
◦
The effect of pH on the enzyme stability was determined by incubating immo-
◦
bilized and free lipase to different pH values ranging from 3 to 12 for 1 h at 4 C,
by using different buffers: glycine–HCl 50 mM (pH 3–4), sodium acetate 50 mM (pH
5
1
8
–6), phosphate 50 mM (pH 7), Tris–HCl 50 mM (pH 8–9) and boric acid 50 mM (pH
0–12). Then the lipase activity was measured under the standard conditions (pH
.5, 37 C). Residual activities were calculated as the ratio of the activity of enzyme
2
.2. Support
◦
Silica aerogels were provided by the Laboratory of Industrial Chemistry II (ENIS),
measured after incubation to the maximal activity of the enzyme.
they were produced as described by Marzouk et al. [18]. Aerogels was obtained by
attack of sol with a solution of sodium hydroxide. In the obtained sodium metasil-
icate sol SiO2–Na2O, we substitute the sodium ions with ammonium ones using
ions exchange resin. The ammonium metasilicate sol transforms into hydrogel after
destabilization with chloric acid 2 M or acetic acid 2 M. The obtained hydrogel is
2
.6.3. Effect of organic solvents on the stability of immobilized lipase
The effect of organic solvents on the stability of immobilized R. oryzae lipase
(
ROLi) was determined using solvents of polarity (log P) ranging from −0.23 to 3.5.
◦
The immobilized lipase was incubated with the experimental solvent for 1 h at 37 C.
Then, the solvent was removed by centrifugation. The immobilized lipase was dried
under vacuum and then assayed for lipase activity using oil olive emulsion as sub-
strate under standard conditions. The residual activity of the solvent-treated lipase
was expressed as the ratio of the activity of treated immobilized lipase to the activity
of the untreated immobilized lipase.
changed in alcoogel by continuous washing with pure ethanol in a soxhlet during
◦
1
0 days. By hypercritical drying of alcoogels in an autoclave at 350 C, they trans-
form into silica aerogel. The approximate values of the pore size are between 5 and
2
−1
1
4
00 nm, the specific surface area is Sp 110 m
g
and the specific pore volume is Vp
−1
. The silica aerogels are used as solid supports for lipase immobilization.
3
cm
g
2
.7. Esterification assay
2.3. Immobilization of lipase
The esterification reactions were performed in screw-capped flasks with a molar
R. oryzae lipase was produced and partially purified as described by Ben Salah
et al. [19]. At the end of the cultivation period, the mycelium was removed by filtra-
ratio of oleic acid to butanol 1:1 (0.4 mmole of oleic acid and 0.8 mmole of butanol),
450 IU of immobilized lipase dissolved in 4 ml of anhydrous n-hexane. The reaction
mixture was shaken for 8 h at 220 rpm at 37 C in a shaking incubator.
Aliquots of 200 ␮l were withdrawn periodically from the reaction mixture. The
immobilized enzyme was removed by centrifugation at 2000 rpm for 5 min, then
the supernatant residual acids contents were assayed by titration with 0.5 N sodium
hydroxide, using phenolphthalein as an indicator and 2 ml of ethanol as a quenching
agent. The conversion (%) of ester synthesis was calculated based on the conversion
of the acid to ester after a given time.
◦
tion. The supernatant was brought to 65% saturation with solid ammonium sulphate
◦
(
NH4)2SO4 followed by centrifugation for 20 min at 8000 rpm and 4 C. The pellet
was resuspended in 50 mM sodium acetate buffer pH 6 containing 100 mM NaCl
and 2 mM benzamidine. Then, the enzyme solution was centrifuged at 8000 rpm
for 10 min and the supernatant containing the lipase (crude lipase) was used for
immobilization.
Adsorption of crude lipase was carried out in batch by adding to a suspension
containing 1% (w/w) of silica aerogels, lipase dissolved in acetate buffers (pH 6)
◦
containing 13,000 IU/ml. The adsorption was conducted for 1 h at 4 C in a shak-
2.8. Reusability of the immobilized R. oryzae lipase
ing water bath. The lipase-adsorbed onto silica aerogels was washed with 20 ml of
◦
acetone priorly chilled at −20 C, filtered through a Buchner funnel and dried at
The esterification of oleic acid with butanol was conducted under these condi-
tions (butanol/oleic acid molar ratio equal to 1; enzyme amount equal to 450 IU;
hexane volume of 3 ml and reaction time of 8 h) using R. oryzae lipase immobilized
onto silica aerogel. This immobilized ROL was reused many times for consecutive
cycles. At the end of each batch, the immobilized lipase was removed from the reac-
tion medium, washed with n-hexane in order to remove any substrate or product
retained in the support and dried at room temperature. Then, the immobilized lipase
was used again for another reaction cycle using fresh substrates.
◦
room temperature. The immobilized lipase was stored at 4 C until use. The yield
of the immobilized lipase activity was defined as the ratio of the adsorbed activity
recovered at the end of the immobilization period divided by the total soluble lipase
activity initially added to 1 g of the support.
2
.4. Determination of protein concentration
2
.9. Statistical analysis
Protein concentrations were determined as described by Bradford [20] using
1%
bovine serum albumin (E1 = 6.7) as reference.
cm
Experimental results were given as mean value ± SD of three parallel measure-
ments. All statistical analysis were conducted using Microsoft Excel.
2.5. Lipase activity
3. Results and discussion
The activities of the free and immobilized lipase were measured titrimetrically
using a pH-Stat (Metrohm, Herisau, Switzerland), under the standard assay con-
ditions described previously using olive oil emulsion as substrate [21]. Briefly, the
reaction mixture contains 10 ml of emulsified olive oil (1 ml of olive oil and 9 ml of
arabic gum at 10%), 20 ml of distilled water and 50 ␮l of bovine albumin serum 12.5%.
3
.1. Adsorption isotherm of ROL onto silica aerogels
To evaluate the loading capacity of the support, 1 g of silica aero-
◦
The reaction was carried out at 37 C and at pH 8.5. The amount of free fatty acid
gels particles were loaded with different initial protein amounts
4300–11,876 ␮g). As shown in Fig. 1, the adsorbed protein amount
increased as more initial protein amount was loaded onto the
released during hydrolysis was estimated by titration with 0.01 N NaOH solution.
Activity was expressed as units per ml of broth (enzymatic solution). One interna-
tional unit (IU) of lipase activity was defined as the amount of enzyme that catalyses
the liberation of 1 ␮mol of fatty acid from olive oil as substrate per min at pH 8.5
(
−
1
support to reach a maximum value of 10,000 ␮g g which is equiv-
alent to 13,000 IU attesting that the adsorbing support surface is
◦
and at 37 C.

N. Kharrat et al. / Process Biochemistry 46 (2011) 1083–1089
1085
1
1
2000
0000
120
A
Immobilized ROLi
Free ROL
100
80
8
000
000
000
000
0
6
4
2
60
4
2
0
0
0
0
2000
4000
6000
8000
10000 12000 14000
2
0
25
30
35
40
45
50
55
60
Iniꢀal amount of protein (μg/g)
Temperature (°C)
Fig. 1. Adsorption isotherm of ROL onto silica aerogels using different initial amount
1
1
20
00
of protein. Lipase activity was measured by pH-stat using olive oil emulsion as
Immobilized ROLi
Free ROL
B
◦
substrate at pH 8.5 and 37 C.
saturated with protein molecules. The immobilized yield of ROL
to silica aerogels reached 95%. This result shows that silica aerogels
have excellent adsorption properties due to its large specific surface
area (Sp 110 m g ) and high porosity (Vp 4 cm g ) which allow
the adsorption of a high amount of protein. In addition, silica aero-
gels are hydrophobic mesoporous materials with methoxy groups
80
60
40
20
0
2
−1
3
−1
(
–OCH3)x, so the adsorption enzyme-support involves multiple
interactions through hydrogen, ionic and hydrophobic interaction.
Ghamgui et al. [22] reported that the immobilization of the
same lipase onto CaCO3 displayed a similar immobilization yield
20
25
30
35
40
45
50
55
60
65
Temperature (°C)
(
93.75%). However, Karra-Chaâbouni et al. [23] reported that the
same R. oryzae lipase immobilized on cellulose fibers, displayed an
immobilization yield of about 70%.
Fig. 3. Effect of temperature on the (A) activity and (B) stability of the free and
immobilized ROL. Enzymes activities were assayed at pH 8.5 using 10 ml of olive oil
emulsion as substrate, 20 ml of distilled water and 50 ␮l of bovine albumin serum
12.5%. Enzymes were incubated 1 h at different temperatures for stability study.
3.2. Adsorption kinetic of ROL onto silica aerogels
The immobilization yield was measured at different incubation
time of an enzymatic solution (13,000 UI) with 1 g of support at
C. As shown in Fig. 2, a 60 min of incubation time of the enzy-
The immobilization onto silica aerogels was carried out with
−
1
13,000 IU g of support during 60 min and gave rise to the high-
est immobilization yield (95%). This result could be explained by
the presence of micropores of the silica aerogels that protect the
lipase against alterations of the microenvironment. Gao et al. [9]
have reported that the immobilization yield of C. rugosa lipase
on methyl-modified silica aerogels by physical adsorption is only
56.4%.
◦
4
matic solution with the support was considered ideal to reach the
maximum adsorption of the enzyme onto silica aerogels (Fig. 2).
A decrease of the adsorbed activity was observed after 120 min of
incubation. This desorption is probably due to the disruption of
the weak physical forces linking the enzyme to its support. Basri
et al. [24] showed that the best immobilization yield of Candida
rugosa lipase onto Amberlite XAD7 was obtained after 30 min of
incubation.
3.3. Biochemical properties of free and immobilized lipase
3.3.1. Effect of the temperature on lipase activity and stability
The immobilization of lipases onto solid support contributes
1
1
1
1
6000
4000
2000
0000
to increase their thermostability and to extend their biotechnol-
ogy potential, since running bioprocesses at elevated temperature
is advantageous because of higher diffusion rate, lower substrate
viscosities, increased reactant solubilities and reduced risk of
microbial contamination [25].
The temperature dependence of the lipolytic activity of free or
immobilized ROL was studied at pH 8.5 in the range of 25–50 C.
8
000
000
000
000
0
◦
6
4
2
The activity profiles of free and immobilized lipase at different tem-
perature are represented in Fig. 3A. The optimum temperature for
◦
both free and immobilized lipases was found to be 37 C. Beyond
◦
40 C, the relative activity of the free lipase declined rapidly, while
the immobilized lipase seemed to be more stable to heat. Indeed,
◦
the immobilized lipase retained 50% of its activity at 45 C and the
0
20
40
60
80
100
120
140
soluble one retained only 20% of its activity and was completely
Incubaꢀon ꢀme (min)
◦
inactivated at 55 C. Silica aerogels may be protecting the enzyme
against thermal denaturation. Similar result was reported in our
previous study, we did not notice changes of the optimum temper-
Fig. 2. Kinetic of ROL adsorption on the silica aerogels. The activity was measured
using olive oil emulsion as substrate at pH 8.5 and 37 C.
◦

1
086
N. Kharrat et al. / Process Biochemistry 46 (2011) 1083–1089
ature for the ROL immobilized onto oxidized cellulose fibers, and
in the same time a significant improvement of its thermal stability
was observed [23]. However, Kılın c¸ et al. [26] have noted a change
in the optimum temperature of porcine pancreatic lipase activity
after its immobilization by adsorption on chitin and chitosan and
subsequent cross-linking with glutaraldehyde.
120
100
80
A
Immobilized ROLi
Free ROL
Thermal stability of soluble and silica aerogels-immobilized ROL
was determined by measuring the residual activity as function
of temperature in the range of 25–60 C (Fig. 3B). Immobilized
60
◦
40
enzyme was inactivated at a much slower rate than the free enzyme
◦
after incubation at 60 C for 1 h, in this condition, the immobi-
20
lized lipase retained 83% of its activity while the free lipase was
completely inactivated. These data indicate that the thermal sta-
bility of lipase was enhanced by the described immobilization
method. This could be due to the lipase location inside the sup-
port micropores which offer a good protection against alterations.
Abdul Rahman et al. [27] found that the thermal stability of C.
rugosa lipase was enhanced after its immobilization onto natu-
ral kaolin by physical adsorption method. De oliveira et al. [28]
have showed that the C. rugosa lipase immobilized on styrene-
divinylbenzen have lost 50% of its activity after only 1 h of heat
0
7
8
9
10
11
12
pH
1
1
20
00
B
Immobilized ROLi
Free ROL
8
6
4
2
0
0
0
0
◦
treatment at 60 C. Under the same conditions, the free enzyme
lost 94% of its activity [28]. It is often found that immobilized
enzyme has a higher thermal stability than free enzyme due to
restriction of its conformational flexibility attributed to its multiple
attachment point of enzyme on the support which limit the confor-
mational alterations and movements under various temperatures
[
29].
0
0
2
4
6
8
10
12
14
3
.3.2. Effect of pH on lipase hydrolytic activity and stability
The effect of pH on the immobilized enzyme activity depends
pH
on the enzyme, the immobilization method and the carrier used.
Effect of pH (7.5–11) on the activity of free and immobilized lipase
was studied. As shown in Fig. 4A, the immobilization did not cause a
shift on the optimal pH for the activity, in fact the highest enzyme
activity was obtained for both forms of the enzyme at pH 8–8.5.
Beyond pH 9, we noted a decrease in the activity for free and immo-
bilized enzyme. Usually the immobilization of enzyme on a cationic
carrier cause a shift on the optimum pH to the acidic range, while,
the immobilization on an anionic carrier cause a shift to the basic
pH values [30]. In general, the immobilized enzymes have either
a broader or the same pH range of high activity than free enzyme
Fig. 4. Effect of pH on the (A) activity and (B) stability of the free and immobilized
ROL. Enzymes activities were assayed at 37 C using 10 ml olive oil emulsion as
substrate, 20 ml of distilled water and 50 ␮l of bovine albumin serum 12.5%. Enzymes
were incubated 1 h at different pH for stability study.
◦
a given solvent between water and octanol in a two-phase system
[39].
Fig. 5 shows that the hexane and the tert-butanol are tolerated
by the silica aerogels-immobilized ROL. Indeed, ROLi maintained
100% of its maximal activity after a long incubation period. As
against, the ROLi looses 50% of its activity in the presence of diox-
ane after 150 min of incubation. Also, we found that the inhibition
is less pronounced when the solvent is more hydrophilic. This could
be explained by the fact that hydrophilic solvents such as acetoni-
[
31–33].
The ROL immobilized onto silica aerogels remained stable and
kept about 95% of its activity within a large range of pH varying from
to 9 (Fig. 4B). The free enzyme looses 60% of its activity at pH 5
5
and 30% of its activity at pH 9. This result indicates that the immo-
bilization procedure improve the lipase stability over a broader pH
range. This result is consistent with other works [34–36]. Krajewska
et al. [37] showed that the immobilization of urease significantly
improved its stability to pH.
1
1
20
00
8
0
3.3.3. Effect of organic solvents on lipase stability
Several organic solvents have various physicochemical effects
60
on enzymes. It is well known that the enzyme activity is strongly
affected by the nature and the polarity of the organic solvent which
may cause the denaturation of the enzyme and thus leads to the
loss of its catalytic activity. The organic solvents change the native
conformation of the enzyme by disrupting of the hydrogen bond
and hydrophobic interactions, leading to alterations of activity and
stability [38]. In this study, the tolerance of immobilized lipase to
organic solvent was studied. Residual activity was measured versus
time in different organic solvents (polar and non-polar) with polar-
ity index (log P) ranging from −0.23 to 3.5 (Fig. 5). The log P value
of a solvent is the parameter used to describe its polarity and its
possible effects on enzyme activity. P is the partition coefficient of
4
2
0
0
Hexane (Log P= 3.5)
Tert butanol(Log P= 0.4)
Acetone (Log P= 0.23)
Acetonitrile(Log P= 0.3)
Dioxane (Log P= 0.42)
0
0
50
100 150
200
250
300
350
400
450
Incubaꢀon Time (min)
Fig. 5. Effect of organic solvents on the stability of immobilized ROL. Enzymes activ-
ities were assayed with olive oil emulsion as substrate at pH 8.5 and at 37 C. The P
value of each used organic solvent is shown.
◦

N. Kharrat et al. / Process Biochemistry 46 (2011) 1083–1089
1087
1
1
20
00
120
Immobilized ROLi
Immobilized ROLi
Free ROL
Fr ee ROL
100
8
0
0
0
0
80
60
40
20
0
6
4
2
0
0
30
60
90
120
150
180
210
240
270
Storage duraꢀon (Days)
0
2
4
6
8
10
Time (h)
Fig. 6. Residual activity of free and immobilized ROL as affected by incubation at
◦
Fig. 7. Synthesis of n-butyl oleate by the free and the immobilised ROL. Reaction
4
C after a storage period of 240 days.
conditions are the following: 450 IU of lipase, 4 ml hexane, an butanol/oleic acid
◦
molar ratio of 1 at 37 C and stirred at 220 rpm.
trile and dioxane can remove the water layers that hydrate the
enzyme preparation and thus distort the catalytic conformation
of the enzyme [40]. These results are on line with several stud-
ies showing the ability of many lipases to remain active exclusively
in the presence of hydrophobic solvents [41].
3
.4.2. Effect of the repeated use of immobilized ROL
The reuse of the enzyme constitutes the main advantage of the
process of biocatalysts immobilization. The immobilized enzyme
is an important parameter for repeated applications in batch reac-
tors or continuously. The reusing of the lipase for several reactions
allowed the reduction of the reaction cost and making an economi-
cally feasible process. Moreover, the use of an immobilized enzyme
permits to greatly simplify the design of the reactor and the control
of the reaction [46]: the filtration of the enzyme stops the reaction;
it is possible to use any kind of reactor, etc. However, the idea of
enzyme reuse implicitly means that the stability of the final enzyme
preparation should be high enough to permit this reuse [3].
3.3.4. Effect of storage temperature on lipase activity
The ability to be stored for a period of time at a certain temper-
ature is an important parameter that must to be considered when
using immobilized lipases. The immobilization of enzyme to a sup-
port often limits its freedom to undergo drastic conformational
changes, and hence results in increased stability towards denat-
uration [29]. Silica aerogels-immobilized lipase retained 100% of
◦
its catalytic activity when stored at 4 C whereas the activity of
To check this parameter, silica aerogels-adsorbed lipase was
used in subsequent cycles in the esterification reaction of oleic
acid with n-butanol under the same experimental conditions as
described above and using 450 IU of lipase. At the end of each batch,
the immobilized enzyme was removed from the reaction medium
and washed with n-hexane in order to remove any substrate or
product retained in the support. Then, the immobilized lipase was
consecutively reused after each reaction cycle. As shown in Fig. 8,
we noted that immobilized ROL on silica aerogels was reused 11
times with the same efficiency (conversion yield of 80%), starting
from the cycle number 12, the conversion yield begin to decrease.
This result show that the immobilization of ROL by adsorption on
silica aerogels allowed not only a good summary of activity in the
the free lipase decreased to 50% after a storage period of 150 days
(
Fig. 6). Moreover, the decrease of the activity over time is more
pronounced in the case of the free ROL which retained only 40%
of its activity after 5 months, while immobilized R. oryzae lipase
remained stable and active during the same period and kept up
to 80% of its activity after 8 months of storage. This confirms that
the silica aerogels provide better stability to the enzyme, due to
its porosity, which protects the three-dimensional conformation of
the lipase and the active site of any distortion or structural changes
that can affect its catalytic activity. In addition, multiple attach-
ments of the enzyme to the support, preventing any intermolecular
process such as proteolysis and aggregation and therefore, creating
a more rigid enzyme molecule [42].
3.4. Esterification activity of the immobilized ROL
100
9
8
7
0
0
0
After immobilization of ROL on silica aerogels, we tested its abil-
ity to catalyze esters synthesis in organic microaqueux media. We
took the synthesis of butyl oleate by direct esterification of butanol
and oleic acid in hexane, as a reaction model. This ester is used in
the field of oleochemistry biodiesel as an additive and as a poly
vinyl chloride plasticizer.
60
5
4
3
2
0
0
0
0
3
.4.1. Kinetic synthesis of butyl oleate with immobilized ROL
As seen in Fig. 7, a high conversion yield of 80% was obtained
with the immobilized lipase. Meanwhile, the free lipase allowed
only 35% of conversion yield. So we have shown not only that
the immobilization of ROL onto silica aerogels improved its activ-
ity and its thermal stability but also increased the performance of
this enzyme to catalyze reactions in a synthesis organic uncon-
ventional media. Several studies have described the production of
esters using various immobilized lipases but the conversion yield
varied considerably with strain [43–45].
10
0
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18
Cycles number
Fig. 8. Effect of repeated use of immobilized ROL on the conversion rate of
butyl oleate. Reaction conditions are following: 450 IU of lipase, 4 ml hexane, an
butanol/oleic acid molar ratio of 1 at 37 C and stirred at 220 rpm. The incubation
time was 8 h for each cycle.
◦

1
088
N. Kharrat et al. / Process Biochemistry 46 (2011) 1083–1089
presence of organic solvent but also the reuse of the enzyme in sev-
eral catalytic cycles. The reuse of the same lipase immobilized on
calcium carbonate and oxidized cellulose was carried out during
the synthesis of butyl oleate and allowed to perform 6 cycles and
[9] Gao S, Wang Y, Wang T, Luo G, Dai Y. Immobilization of lipase on
methyl-modified silica aerogels by physical adsorption. Bioresour Technol
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[
[
10] Novak Z, Habulin M, Krmelj V, Knez Zˇ . Silica aerogels as supports for lipase
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2003;27:169–78.
3
cycles, respectively [22,23]. Blanco et al. [47] showed that the
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lipase from C. antarctica B immobilized on the mesoporous silica
allowed a quantitative conversion even after 15 reaction cycles.
Two commercial lipases (Burkholderia cepacia and C. antarctica)
were encapsulated in silica aerogels. These immobilized lipases
were applied in biodiesel synthesis by transesterification of sun-
flower seed oil with methyl acetate, the reuse of these lipases
allowed 11 cycles and kept only 60% of their initial activity [48].
The activity of ␣-chymotrypsin immobilized to magnetic particles
did not significantly change after 12 repeated uses during the pro-
teolytic cleavage of porcin pepsin [49].
2003;26:47–56.
[
[
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4
. Conclusion
The key step in the enzymatic process consists on the successful
immobilization of the enzyme allowing its recovery and reuse. The
effectiveness of an immobilization process depends on the support
used. This work focuses on the immobilization of ROL onto silica
[
aerogels by physical adsorption. The immobilization carried out at
◦
4
C during 60 min gave rise to the highest immobilization yield
[19] Ben Salah R, Fendri K, Gargouri Y. La lipase de Rhizopus oryzae: pro-
(
95%). The silica aerogels-immobilized lipase displayed a better sta-
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bility than the free form. Indeed, immobilization of the enzyme
enhanced its stability towards to the temperature and pH. More-
over the immobilized enzyme was shown to be well tolerant to
apolar solvent like hexane and improved storage stability.
[
[
20] Bradford A. Rapid and sensitive method for the quantitation of microgram
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In order to evaluate the efficiency of immobilized enzyme for the
esterfication reaction, we used oleic acid and butanol as reagents
in hexane solvent. Our results showed that the highest yield was
◦
obtained at 37 C with a molar ratio of oleic acid to butanol 1:1
[
[
[
and 450 IU of immobilized lipase. This system could be reused 11
times without a significant loss of the activity. Therefore, the silica
aerogels may have a promising future as support for biocatalysts in
various syntheses.
Acknowledgements
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This work is part of a doctoral thesis by Nadia KHARRAT. This
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