Esterification activity and stability of Talaromyces thermophilus lipase immobilized onto chitosan

Article abstract of DOI:10.1016/j.molcatb.2010.11.010

The Talaromyces thermophilus lipase (TTL) was immobilized by different methods namely adsorption, ionic binding and covalent coupling, using various carriers. Chitosan, pre-treated with glutaraldehyde, was selected as the most suitable support material preserving the catalytic activity almost intact and offering maximum immobilization capacity (76% and 91%, respectively). The chitosan-immobilized lipase could be reputably used for ten cycles with more than 80% of its initial hydrolytic activity. Shift in the optimal temperature from 50 to 60 °C and in the pH from 9.5 to 10, were observed for the immobilized lipase when compared to the free enzyme. The catalytic esterification of oleic acid with 1-butanol has been carried out using hexane as organic solvent. A high performance synthesis of 1-butyl oleate was obtained (95% of conversion yield) at 60 °C with a molar ratio of 1:1 oleic acid to butanol and using 100 U (0.2 g) of immobilized lipase. The esterification product is analysed by GC/MS to confirm the conversion percentage calculated by titration.

Full text of DOI:10.1016/j.molcatb.2010.11.010

Journal of Molecular Catalysis B: Enzymatic 68 (2011) 230–239
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Esterification activity and stability of Talaromyces thermophilus lipase
immobilized onto chitosan
Ines Belhaj-Ben Romdhane, Zamen Ben Romdhane, Ali Gargouri, Hafedh Belghith∗
Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologies de Sfax, BP “1177” 3018 Sfax, Tunisia
a r t i c l e i n f o
a b s t r a c t
Article history:
The Talaromyces thermophilus lipase (TTL) was immobilized by different methods namely adsorption,
ionic binding and covalent coupling, using various carriers. Chitosan, pre-treated with glutaraldehyde,
was selected as the most suitable support material preserving the catalytic activity almost intact and
offering maximum immobilization capacity (76% and 91%, respectively). The chitosan-immobilized lipase
could be reputably used for ten cycles with more than 80% of its initial hydrolytic activity. Shift in the
optimal temperature from 50 to 60 ◦C and in the pH from 9.5 to 10, were observed for the immobilized
lipase when compared to the free enzyme.
Received 15 July 2010
Received in revised form
22 November 2010
Accepted 22 November 2010
Available online 27 November 2010
Keywords:
The catalytic esterification of oleic acid with 1-butanol has been carried out using hexane as organic sol-
vent. A high performance synthesis of 1-butyl oleate was obtained (95% of conversion yield) at 60 ◦C with
a molar ratio of 1:1 oleic acid to butanol and using 100 U (0.2 g) of immobilized lipase. The esterification
product is analysed by GC/MS to confirm the conversion percentage calculated by titration.
© 2010 Elsevier B.V. All rights reserved.
Talaromyces thermophilus lipase
Immobilization
Chitosan
Esterification
1. Introduction
lipase and its carrier matrix and allowed its reuse more often than
other available immobilization methods such as adsorption and
Lipases have gained a great interest in several biotechnology
applications due to their broad specificity coupled with high enan-
tio and regio selectivity. This interest arises from the ability of these
enzymes to catalyze synthetic reactions occurring in non aqueous
media. In fact, lipase have been employed for direct esterifica-
tions and transesterifications reactions in organic media to produce
esters used as additives for a variety of perfumes and flavours [1],
biosurfactants [2] and biofuels [3]. The main hurdle to the use of
lipase for these applications is the cost of biocatalysts. One of the
principal goals of immobilization procedures is to fix the maximum
amount of enzymes by keeping maximum activity at the lowest
cost. Lipase immobilization onto solid materials not only enhances
the operational lifetime and stability of biocatalysts but also facili-
tates the recovery, reuse and continuous operation of lipases. It may
also protect the enzyme from solvent denaturation and enhance the
enzyme thermostability [4].
entrapment [11]. Inorganic and organic supports have been suc-
cessfully used for the immobilization of enzymes. Although there is
no universal support still suitable for all enzymes and all their appli-
cations, any material that is to be considered as enzyme support
must fulfil some requirements: high affinity for proteins, avail-
ability of reactive functional groups, mechanical stability, rigidity,
feasibility of regeneration, non toxicity and biodegrability [12].
However, the high cost of the materials that are commonly used
for lipase immobilization (namely, silica-based carriers, acrylic
resins, synthetic polymers, active membranes, exchange resins,
etc.) as well as the technology necessary to apply fixation methods
greatly increase the costs of biocatalysts. Some cheaper substitutes,
exhibiting high activity and stability to the immobilized lipases,
have been widely used, such as rice husk, rice straw [13], amberlite
[14], chitin and chitosan [15].
Biotechnology production of esters from long chain fatty acids
using immobilized lipases has recently received greater considera-
tion over the traditional chemical synthetic methods. Among these
esters, 1-butyl oleate is used as biodiesel, poly vinyl chloride plas-
ticizer, water-resisting agent and hydraulic fluid [16,17]. The most
desired characteristics of lipases used for this process, are its abil-
ity to utilize all mono, di, and triglycerides as well as the free fatty
acids in transesterification, low product inhibition, high activity
and yield in non-aqueous media, low reaction time, reusability of
immobilized enzyme, temperature, solvent resistance and alcohol
resistance [18,19]. Various lipases from different sources, immo-
Several approaches have been reported for the immobiliza-
tion of lipases, they consisted either on physical adsorption of the
enzyme on a carrier material [5,6], its entrapment or microencapsu-
lation in a solid support [7,8] or by covalent binding to a solid matrix
[9,10]. Immobilization using covalent binding has been most widely
studied. Covalent binding provides a powerful link between the
∗
Corresponding author. Tel.: +216 74874449; fax: +216 74874449.
E-mail address: hafeth.belghith@cbs.rnrt.tn (H. Belghith).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.11.010

I.B.-B. Romdhane et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 230–239
231
bilized on different carriers, have shown a range of yields with
different oil substrates and different acyl acceptors. The most previ-
ously reported reactions of the 1-butyl oleate production have been
performed by commercial lipases such as from Candida rugosa [20],
Rhizomucor miehei [21,22] and Pseudomonas fluorescens [23,24].
A search for an ideal enzyme with more suitable properties
remains important. In this present investigation, we are developed
an appropriate method to immobilize a new purified lipase from
a thermophilic fungus Talaromyces thermophilus (TTL). The stabil-
ity of this immobilized lipase and its suitability for reuse were
evaluated. Since the T. thermophilus fungus produces high stable
xylanolytic activities, the production of fermentable sugar from
lignocellulosic material waste was previously undertaken [25]. Fur-
ther studies are currently under way to achieve the bioethanol
production. This present contribution established another alter-
native exploring the lipolytic activity of this fungus for produce
a biodiesel (butyl oleate) as a valuable starting material for further
selective enzymatic synthesis of oleyl alkyl esters using different
oil sources.
at 1.7 void volume) were pooled, concentrated and injected to
the FPLC column Mono-Q Sepharose (trimethylammonium anion
exchange) equilibrated in buffer A. The column (1 cm × 10 cm) was
rinsed with 40 ml of the same buffer. Adsorbed material was eluted
with a linear gradient of 100–400 mM NaCl in the same buffer at
a rate of 120 ml h−1. TTL activity was eluted at 220 mM NaCl. The
highly active lipase fractions were pooled, concentrated and used
as purified enzyme for subsequent studies. The purified enzyme
has a high specific activity reached 9800 139 U mg−1 using olive
oil emulsion as substrate in the presence of 2 mM CaCl2 at pH 9.5
and 50 ◦C [27].
2.4. Lipase immobilization
T. thermophilus lipase was immobilized by different methods on
different supports. The immobilization steps and enzyme storage
were carried out at 4 ◦C. The supernatants and washing volumes
were pooled after each step and non immobilized activity was
determined.
2. Materials and methods
2.4.1. Adsorption
Adsorption followed by precipitation (CaCO3): The enzyme immo-
bilization was made onto CaCO3 according to Rosu et al. [28] with
a slight modification. A support powder (1 g) was added to 2 ml of
enzymatic solution (285 U/ml, 29 ␮g/ml). The mixture was incu-
bated 1 h at 4 ◦C under mild agitation. Afterwards, 10 ml of chilled
acetone were added and the suspension was filtered through a
Buchner funnel. The preparation of immobilized lipase was washed
two times with 20 mM Tris–HCl buffer, centrifuged for 2 min at
4500 × g, dried at room temperature and stored at 4 ◦C until use.
Ionic binding (Amerlite IRC-50, Duolite, DEAE-Sephadex)
2.1. Chemicals
Chitosan flaks form (having a deacetylation degree of 85%
and a bulk density of 0.15–0.3 g/cm3) was provided from Sigma,
chitin (powder form) was obtained from Sigma, glutaraldehyde
and Gelatin were from Amersham. Amberlite IRC-50 and Duolite
C26 were provided from Rohm and Haas (France). DEAE-Sephadex
was from Pharmacia. Arabic gum from Merck (D-6100 Darm-
stadt, Germany); oleic acid, hexane and 1-butanol were purchased
from Prolabo (Paris, France); pH-stat (718 Stat Titrino) was from
Metrohm (Herisau, Switzerland).
•
The Amberlite IRC-50 or Duolite were swollen in distilled water,
and equilibrated with 20 mM Tris–HCl buffer. One gram of each
of the suction-filtered support material was mixed with 2 ml of
enzyme solution (285 U/ml, 29 ␮g/ml). The mixture was intermit-
tently stirred with a glass rod for 5 h at 4 ◦C. Then, it was washed
twice with an equal volume of 20 mM Tris–HCl buffer, centrifuged
for 2 min at 4500 × g, dried at room temperature and stored at 4 ◦C
until use.
2.2. Microorganism and culture conditions
The present study reports on a newly isolated thermotolerant
fungal strain isolated from a soil sample collected in the thermal
station of El Hamma in the south of Tunisia. The fungal isolate
was identified as T. thermophilus by CBS (Centraalbureau voor
schimmelculturen, Holland Code reference: detail 274-2003). The
T. thermophilus strain was also deposed in a national strain bank of
Tunisia: Tunisian Collection of Microorganisms CTM10.103 (Cen-
tre of Biotechnology of Sfax, Tunisia) [25]. The T. thermophilus was
cultivated in a modified liquid Mandels medium [26]. The basal
medium contained KH2PO4 1 g/l, K2HPO4 2.5 g/l, (NH4)2SO4 1.4 g/l,
MgSO4·7H2O 0.3 g/l, CaCl2 0.3 g/l, yeast extract 1 g/l, urea 0.7 g/l,
Tween 80 1 ml/l and 1 ml/l of an oligoelement solution (MnSO4:
1.6 g/l, ZnSO4: 1.4 g/l, FeSO4: 5 g/l, CoCl2: 2 g/l). Wheat bran at 2%
was added to one litre of this medium, as a carbon source.
The enzyme production was carried out in 500 ml flasks con-
taining 100 ml of culture medium that was incubated at 50 ◦C at an
agitation rate of 160 rpm for 4 days.
•
1 g resin DEAE-Sephadex was washed twice with 20 mM Tris–HCl
buffer pH 9 and centrifuged for 2 min at 4500 × g. The resin was
mixed with 2 ml of the enzyme preparation (285 U/ml, 29 ␮g/ml)
and 2 ml Tris–HCl buffer for 20 min under agitation. The mix-
ture was then washed twice with 20 mM Tris–HCl buffer and
centrifuged for 2 min at 4500 × g.
2.4.2. Covalent coupling (chitosan, chitin and gelatin)
The chitosan (0.5 g) was completely dissolved in 50 ml of 0.1 M
•
HCl, after continuous agitation for 24 h at 30 ◦C. Then, a glu-
taraldehyde solution of 1.5% (v/v) was added for 2 h at the same
temperature. The solubilized chitosan was precipitated by the
addition of 1 ml NaOH (1 M). The precipitate was separated by
centrifugation (10 min at 4500 × g) and washed with distilled
water to remove excess of glutaraldehyde, which did not react
with the active groups of the support, to ovoid the reticulation of
the enzymes molecules; affecting therefore their activities [29].
2.3. Enzyme preparation protocol
The extracellular proteins were recovered by centrifugation and
the supernatant was treated with ammonium sulfate (60% satura-
tion). The precipitate was collected by centrifugation at 9000 rpm
for 30 min, dissolved in buffer A (20 mM Tris–HCl, pH 8.5; 20 mM
NaCl and 1 mM benzamidine). Insoluble material was removed by
centrifugation at 9000 rpm during 10 min. The enzyme solution was
loaded on a column (3 × 160 cm) of gel filtration Sephacryl S-200
equilibrated with buffer A.
The wet chitosan was mixed with 1 ml (285 U/ml) of the enzyme
solution and stirred at 4 ◦C for 24 h. The unbound enzyme was
removed by washing with Tris–HCl buffer 20 mM until no protein
or activity was detected [30]. This protocol was repeated in the
presence of different glutaraldehyde concentrations (0.5–3%) (v/v)
to investigate the latter’s effect on the immobilization and activity
yields of the enzyme.
The elution of lipase was performed with the same buffer at a
rate of 45 ml h−1. The fractions containing the lipase activity (eluted

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•
Chitin (0.5 g) was shaken with 5 ml 2.5% (v/v) glutaraldehyde.
of proteins was calculated as following:
It was then collected by centrifugation (10 min at 4500 × g) and
ꢀ
ꢁ
Amount of adsorbed proteins
Initial amount of proteins
washed with distilled water to remove excess of glutaraldehyde.
The wet chitin was mixed with 2.0 ml of the enzyme solution at
4 ◦C for 24 h. The unbound enzyme was removed by washing with
Tris–HCl buffer 20 mM as described earlier [30].
Proteins loading (%) =
× 100
2.7. Scanning electron microscopy (SEM)
•
The gelatin powder (5%, w/v) was swelled in 5 ml of Tris–HCl
buffer (50 mM, pH 9.0) and heated at 50 ◦C for 10 min to ensure
its complete solubilization. The mixture was then cooled and the
enzyme solution was added. After the thorough mixing of the
enzyme, the required amount of organic cross-linker (0.6%, w/v)
glutaraldehyde was added. The mixture was constantly stirred
and then poured on a (5 cm × 5 cm) glass plate to prepare a thin
film of the enzyme. The film was stored at 4 ◦C for complete cross-
linking. The immobilized enzyme film was thoroughly washed
with 20 mM Tris–HCl buffer (pH 9.0) and cut into small blocks
before being used in subsequent experiments.
A Baltec Critical Point Dryer 30 field emission scanning electron
microscope (Balzers Union, Germany) equipped with a secondary
electron detector and energy dispersive X-ray microanalysis (EDX)
was used to examine the surface morphology of the carrier before
and after the immobilization of TTL on chitosan. The samples were
sputter-coated with gold prior to analysis.
2.8. Characterization of immobilized lipase
2.8.1. Effect of pH on the free and the immobilized lipases
activities and stabilities
The effect of pH on free and immobilized lipase activities was
studied in the pH range 7.5–11 at 50 ◦C using olive oil as substrate.
Each measurement was performed three times and standard error
was included.
The immobilization was estimated as following:
A − B
Immobilization yield (%) =
× 100
A
The enzyme stability was determined by incubation of the
immobilized and free lipase at different pH values ranging from 3
to 11 for 24 h at 4 ◦C using the following 50 mM buffers systems:
Na2HPO4/citrate, pH 3.0–5.0; KH2PO4/K2HPO4, pH 6.0–7.0; 3-N-
(␣,␣-dimethylhydroxyethyl)-amino-2-hydroxypropanesulfonic
acid (AMPSO), pH 8–9; and glycine, pH 10–11 and their residual
activities were determined at the optimum pH (9.5 for the free
enzyme and 10.0 for the immobilized enzyme). Each measurement
was performed three times and standard error was included.
ꢀ ꢁ
C
A
Activity yield (%) =
× 100
with A is the total enzyme activity used for immobilization; B is
the unbound enzyme activity; A − B is the theoretical immobi-
lized enzyme activity; and C is the obtained immobilized enzyme
activity. The total enzyme activity is the total number of units
added to the support during the immobilization reaction; the
non-immobilized activity is the number of units found in filtrates
and washing volumes after immobilization; and the immobilized
activity is the number of units detected in the support after immo-
bilization and washing.
2.8.2. Effect of temperature on the free and the immobilized
lipase activities and stabilities
The effect of temperature on the hydrolytic activities of both
forms of TTL was determined at various temperature values ranging
from 30 to 70 ◦C. Their relative activities were determined at pH 9.5
for the free lipase and at pH 10.0 for the immobilized lipase, using
olive oil emulsion as substrate. Each measurement was performed
three times and standard error was included.
The thermal stability assays were performed by incubation of
the immobilized and free forms of TTL at different temperatures
(30–70 ◦C) for 1 h, cooled down to room temperature and their
residual activities were measured under optimal conditions (pH
9.5 and 50 ◦C for the free lipase; pH 10.0 and 60 ◦C for the immo-
bilized lipase). Each measurement was performed three times and
standard error was included.
2.5. Lipase activity determination
Lipase activity was assayed potentiometrically by automatically
titrating the free fatty acids released from mechanically stirred
triglyceride emulsions at pH 9.5 and 50 ◦C, using 0.1 N NaOH and
a pH-stat device (718 Stat Titrino, Metrohm, Switzerland). The
triglycerides were added directly to the pH-stat vessel contain-
ing the assay solution and were emulsified by mechanical stirring.
Long-chain triglycerides (e.g. olive oil) had first to be pre-emulsified
with gum arabic (GA) by mixing 5 ml of olive oil with 45 ml of a 10%
(w/v) GA solution prepared as previously described [31]. Ten ml of
this olive oil-GA emulsion were then mixed in the pH-stat vessel
with 30 ml of a solution containing 20 mM Tris–HCl, 2 mM CaCl2
[32].
2.9. Reusability of immobilized lipase
The determination of immobilized lipase activity was initiated
by the addition of 0.1 g of support containing the immobilized
enzyme to the substrate. One unit of lipase activity corresponds to
1 ␮mol of fatty acid released per minute under the assay conditions
used.
For the reusability, after each reaction run, the immobilized
lipase preparation was removed and washed with 20 mM Tris–HCl
buffer to remove any residual substrate. It was then reintroduced
into fresh reaction medium to determine enzyme activity. The
experiment was performed three times and standard error was
included.
2.6. Protein assay
2.10. Esterification assay
The protein content in the free enzyme or immobilized enzyme
preparations was determined by the Bradford method, using bovine
serum albumin (BSA) as the standard (the protein is, therefore,
expressed in BSA equivalents) [33]. The amount of protein bounded
onto the support was determined indirectly from the difference
between the initial total protein exposed to the supports and the
amount of protein recovered in the wash. The loading percentage
The reactions were carried out in screw-capped flasks contain-
ing 1 g of oleic acid and butanol taken at various molar ratios, in
the presence of 4 ml of anhydrous hexane. Different amounts of
chitosan immobilized lipase (50 U in 0.1 g); (100 U in 0.2 g); (200 U
in 0.4 g) and (300 U in 0.6 g) and different levels of added water (0%,
2%, 5% and 7%) were tested. The reaction mixture was incubated at

I.B.-B. Romdhane et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 230–239
233
Table 1
Variation in immobilization and activity yields of lipase as function of resin used.
Type of resin
CaCO3
Amerlite
Duolite
DEAE-Sephadex
Chitin
Chitosan
Gelatin
Activity yield (%)
15.0
85.5
ND
24.0
137
32.0
31.0
20.0
114
30.0
29.6
48.0
274
50.4
46.0
65.0
371
88.5
66.2
76.0
433
91.0
70.0
56.0
319
62.6
51.0
Activity (U/g carrier)
Immobilization yield (%)
Immobilized proteins (%)
ND
60 ◦C in a shaking incubator at 200 rpm. Each experiment was twice
repeated.
and the contact time between enzyme solution and these supports
need to be carried out.
Aliquots of 200 ␮l were withdrawn periodically and centrifuged
at 2000 rpm for 5 min. The supernatant residual acids contents were
assayed by titration with 0.1 N sodium hydroxide, using phenolph-
thalein as indicator and 3 ml of ethanol as quenching agent. The
yield of ester synthesis was calculated based on the amount of acid
consumed.
Analyses were confirmed by GC/MS using a GC system equipped
with a series 5975 B Insert MSD mass-selective detector (Agilent
technologies, France). 2 ␮l portion of the organic phase was anal-
ysed after splitless injection employing a HP-5MS Phenyl Methyl
Siloxane capillary column (30 m × 250 ␮m × 0.25 ␮m nominal, Agi-
lent Technologies, France). Helium (constant flow, 1 ml min−1) was
used as a carrier gas. The temperatures of the injector and detector
were 250 ◦C and 240 ◦C, respectively. The following temperature
program was applied: 120 ◦C for 5 min, increase of 3 ◦C min−1 to
180 ◦C, increase of 10 ◦C min−1 to 220 ◦C, and 220 ◦C for 31 min.
Data were evaluated using the NIST Mass Spectral Search program
[34]. The conversion percentage calculated by both GC/MS analysis
(which showed product formation) and titrimetry (which showed
acid consumption) were found to be in good agreement.
Low yields of immobilized enzyme activity were obtained for
all this binding resins, essentially for Amerlite IRC-50 and Duolite
C26 carriers. This could imply that their surfaces are not physi-
cally inert towards lipase. Thus, interaction of lipases with these
supports often leads to structural deformations and to reduced cat-
alytic activity [36]. An additional factor would be a weak interaction
between the lipase and the support. The adsorbed lipase was sen-
sitive to the pH and the ionic strength variations and the protein
might be stripped off from the carrier.
It has been found in many examples that the immobilization
of lipases on hydrophobic supports permits to get a higher activ-
ity recovery of the immobilized preparations [37]. In fact, these
supports involving the adsorption of the hydrophobic areas sur-
rounding the active site and leaving stabilized the lid-opened form
of the lipase [37]. However, if the substrate is very large (e.g. olive
oil), the near presence of the hydrophobic support surface may gen-
erate steric hindrances, reducing largely the activity of the lipase
[38].
Covalent coupling was more advantageous than other, since
diffusional restrictions of substrates and products are decreased
considerably. On the other hand, multipoint covalent liaison via
short spacer arms promotes the stabilization of the immobilized
structure enzyme. In fact, the relative distances among all residues
involved in the covalent immobilization have to be unaltered dur-
ing any conformation change induced by any distorting agent (heat,
extreme pH values, organic solvents, . . .) [38]. This also should
reduce the steric hindrance which restraining the access of the sub-
strate to the lipase active site [39]. TTL was therefore covalently
coupled to chitin, chitosan and gelatin, through cross-linking medi-
ated by glutaraldehyde. Compared to chitin and gelatin, chitosan
provided the highest activity (433 U/g chitosan) and immobiliza-
tion yields (76%). Chitosan is a cheap support material since it
was obtained from prawn’s shells. Moreover, this polyaminosac-
charide was recognized by excellent properties for lipase support,
such as biocompatibility, biodegradability, physiological inertness,
hydrophilic character and great affinity for proteins [15].
Furthermore, this support was selected as a suitable adsorbent
leading to high dispersion of the TTL in the support and preserving
the catalytic activity. Magnin et al. (2001), reported the immobi-
lization of C. rugosa lipase into porous chitosan beads; they found
that immobilization enhanced the lipase activity and its tolerance
to organic solvents [40].
3. Results and discussion
3.1. Study of immobilization support
The lipase from T. thermophilus (TTL) was immobilized on differ-
ent matrix with various methods. Table 1 shows the immobilization
and activity yields of the used matrix and the percentages of bound
proteins.
The lipase adsorption followed by precipitation was carried out
with a non-ionic carrier CaCO3. This support carrier was previously
described as a suitable adsorbent leading to high dispersion of the
crude Rhizopus oryzae lipase and preserving the catalytic activity
[17]. The same support was found to be appropriate for the Pseu-
domonas SP KWI 56 [28]. In contrast to these results, the purified
TTL adsorbed on CaCO3 show a very low yield of the immobilized
lipase activity. It has been reported that the presence of ammonium
sulfate was needed to help the enzyme immobilization on CaCO3
[17,35].
The enzyme adsorption by ionic binding was occurred by three
binding resins: two carboxylic cation exchangers (Amberlite IRC-50
and Duolite C26) and an anionic exchanger (DEAE-Sephadex). The
adsorption on Amberlite IRC-50 and Duolite C26 led to a poor yield
of adsorbed lipase. It is known that the ionic adsorption is based on
electrostatic interactions between differently charged ionic groups
of the matrix and of the enzyme. Also, a weakly hydrogen bonds and
Van Der Waals forces are formed during the adsorption. A repul-
sion force between the negative surface charge of the Amberlite
IRC-50 and Duolite C26 carriers and the negative charged of the
TTL occurred at the pH of the immobilization (pH 9), can lead to the
leaching of enzyme weakly adsorbed, which explains the poor yield
of the immobilized enzyme. This minimally adsorption was hap-
pened only due to the hydrophobic properties of these two carriers.
The anionic DEAE-Sephadex resin performed a moderate enzyme
adsorption. Further investigations to optimize the enzyme loadings
A possible explanation for the low loss of TTL activity is the
potential for glutaraldehyde reactive group to link protein through
amino groups of lysines existing in the lid (data not shown). This
interaction may generate some steric hindrances, avoid the dis-
placement of the lid and then reducing the catalytic action of the
lipase.
3.2. Influence of glutaraldehyde concentration on the
immobilization and activity yields of lipase
Glutaraldehyde is a bifunctional crosslinker commonly used to
couple components with amino functional groups. However, glu-
taraldehyde is toxic and causes the denaturation of immobilized

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Table 2
showing that the immobilization methods preserved the enzyme
activity over a wider pH range. Lipase of Candida lipolytica immo-
bilized on silanized palygorskite support by covalent bonding
retained nearly 100% of activity within a pH range of 6.5–8 whereas
it loses more than 87% of its activity at pH 6.5 in its free state
[44]. Similarly, the lipase from C. rugosa covalently immobilized
onto polyacrylonitrile nanofibers was less sensitive to pH changes
exhibiting the same optimum pH than the free lipase [44].
The stability of the free and immobilized TTL was compared at
pH ranging between 3.0 and 11.0 at 4 ◦C during 24 h of incubation.
At different pH conditions, the immobilized lipase was more stable
than the free enzyme (Fig. 2B). At acid pH (3.0 and 5.0) the immobi-
lized lipase lost 45% of its residual activity, whereas in its free state
this loss was twice higher. This could be attributed to the ability
of the micro-environment, created between the support and the
immobilized enzyme, to protect the latter from the denaturation
caused by the change in pH. This stabilizing effect in acidic and
alkaline media has been previously reported for C. lipolytica immo-
bilized lipase on silanized palygorskite [45]. Then, we can conclude
that the chitosan support has a special importance of this immobi-
lization technique in the lipase stabilization at different ranges of
pH.
Effect of glutaraldehyde concentration on immobilization and activity yields of
lipase.
Glutaraldehyde (GA) concentration (%)
0.5
1
1.5
2
2.5
3
Activity yield (%)
Immobilization yield (%)
57
68
70
84
77
90
65
87
43
82
37.5
70.6
enzymes [41]. Therefore, the effects of glutaraldehyde concen-
tration on the enzyme’s immobilization and activity yields were
investigated (Table 2). The chitosan support was prepared in
the presence of different glutaraldehyde concentrations (0.5–3%)
(v/v) and the immobilization and activity yields were measured.
Results showed that a glutaraldehyde concentration of 1.5% (v/v)
is optimal for lipase recovery reaching 77% of the yield activity.
Similar result was observed for the immobilization of lipase on the
polysulfone membrane surface [42]. At higher glutaraldehyde con-
centrations, more protein is bond; but with lower activity. This may
be due to the steric hindrance occasioned by the increased level
of polymerization in the enzyme-support matrix which impedes
the access of the substrate to the active site of the immobilized
enzyme [43].
3.3. Morphology by scanning electron micrograph (SEM)
3.5. Thermal stability and optimal temperature of immobilized
lipase
The morphology of the chitosan pre-treated with glutaralde-
hyde before and after the immobilization was examined. We
observed that the chitosan activated by glutaraldehyde has a large
surface area (Fig. 1A). After lipase immobilization, the support sur-
face area was filled with rounded structures, which were likely to
be protein aggregates (Fig. 1B). The flat chitosan surface seems to
provide a large contact area for a multipoint attachment with the
enzyme. This observation may offer an explanation for the high
yield of the T. thermophilus lipase immobilization on this support.
Same results were also observed by Foresti and Ferreira after immo-
bilization of C. rugosa lipase onto chitosan [12].
The temperature dependence of the activity of the soluble and
immobilized lipase was studied (at pH 9.5 for the free enzyme and
pH 10.0 for the immobilized enzyme). The free and the immobi-
lized enzyme exhibited different temperature profiles with a shift
of optimal reaction temperature from 50 to 60 ◦C (Fig. 2C). A similar
shift of 10 ◦C was also reported for C. rugosa lipase immobilized on
styrene divinylbenzene copolymer [46].
Thermal stability of the free and the immobilized lipase was fol-
lowed after their incubation at different temperatures ranging from
30 to 70 ◦C for 60 min. As seen in Fig. 2D, the immobilized lipase was
much more stable than the free enzyme particularly when the tem-
perature exceeded 50 ◦C. Thus, after heat treatment for 60 min at
70 ◦C, more than 50% drop of the initial activity is observed for the
free lipase whereas it attains only 22% for the immobilized enzyme.
This result clearly demonstrates the efficiency of the immobiliza-
tion method in the enzyme protecting against heat inactivation.
It could be the consequence of conformational limitations of the
enzyme movements due to multi-point attachment and it is in
agreement with other reported works [47,48]. The better thermal
stability of the immobilized will extend the potential application
of the lipase as a biocatalyst.
3.4. pH stability and optimum pH of immobilized lipase
The effect of pH on the activity of both free and chitosan immo-
bilized lipase on olive oil hydrolysis was determined in the pH
range of 7.5–11 and the results were presented in Fig. 2A. The
pH profile seems to be identical for the free and the immobilized
enzyme. Furthermore, the optimum pH shifted from pH 9.5, which
was the optimum for the free enzyme, to more alkaline range
(pH 10).
The retained activity of the immobilized enzyme was improved
both at lower and higher pH in comparison to the free enzyme,
Fig. 1. Scanning electron micrographs of the chitosan (A) before (950×) and (B) after the immobilization of TTL (1200×).

I.B.-B. Romdhane et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 230–239
235
120
120
100
80
60
40
20
0
C
A
100
80
60
40
Free lipase
20
0
Free lipase
Immobilized lipase
Immobilized lipase
20
30
40
50
60
70
80
6
7
8
9
10
11
Temperature (°C)
pH
120
B
120
100
80
60
40
20
0
D
100
80
60
40
20
0
Immobilized lipase
Free Lipase
Free lipase
Immobilized lipase
0
2
4
6
8
10
12
20
40
60 80
Temperature (°C)
pH
Fig. 2. (A) Effect of pH on free (ꢀ) and immobilized (ꢁ) T. thermophilus lipase activities. Enzymes were assayed with olive oil emulsion as substrate at 50 ◦C. (B) The effect of
pH on the stability of free (ꢀ) and immobilized (ꢁ) T. thermophilus lipase. Enzymes were assayed with olive oil emulsion as substrate at 50 ◦C. Bars indicate standard deviation.
(C) Effect of temperature on free (ꢀ) and immobilized (ꢁ) lipase activities. Enzymes were assayed with olive oil emulsion as substrate at pH 9.5 and pH 10, respectively. (D)
Thermal stability of free (ꢀ) and immobilized (ꢁ) TTL. Enzymes were assayed, after incubation for 1 h at different temperatures, with olive oil emulsion as substrate at optima
conditions. Bars indicate standard deviation.
3.6. Reusability
As shown in Fig. 4, the immobilized enzyme displayed a good
reusability, since it retained more than 80% of its initial hydrolytic
activity in reuse up to 10 cycles. This finding is of a great inter-
est, as it would allow the set-up of a bioreactor for continuous
application.
Chitosan immobilized lipase was tested repeatedly to hydrolyze
olive oil at 60 ◦C and the reusability was examined because of its
importance in batch and in continuous reactor for bioconversion
processes.
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0
2
4
6
8
10
12
Cycle number
Fig. 3. Effect of organic solvents on the activity of immobilized TTL. Enzyme activ-
ities were assayed, after 2 h of incubation with different solvents, using olive oil as
substrate at pH 10.0 and 60 ◦C. The experiments were conducted three times and
standard errors are reported.
Fig. 4. Effect of repeated use on the olive oil hydrolysis by the immobilized TTL at
60 ◦C and pH 10.0. The experiments were conducted three times and standard errors
are reported.

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I.B.-B. Romdhane et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 230–239
Fig. 5. (A) Effect of different amounts of immobilized TTL: (♦) 50 U, (ꢁ) 100 U, (ꢂ) 200 U and (ꢀ) 300 U, on the conversion yield during butyl oleate synthesis. Reaction
conditions were oleic acid/butanol molar ratio of 1/1 at 60 ◦C and stirred at 250 rpm. Bars indicate standard deviation. (B) Effect of different initial added water: (ꢀ) 0%, (ꢁ)
2%, (ꢂ) 5% and (♦) 7%, on the conversion yield during butyl oleate synthesis. Reaction conditions were 100 U of lipase, an oleic acid/butanol molar ratio of 1/1 at 60 ◦C and
stirred at 250 rpm. Bars indicate standard deviation. (C) Effect of different oleic acid/butanol molar ratios (R): (ꢀ) 1/1, (ꢁ) 1/2, (ꢂ) 1/3, (♦) 2/1 and (ꢃ) 3/1, on the conversion
yield during butyl oleate synthesis. Reaction conditions were 100 U of lipase; 0% added water at 60 ◦C and stirred at 250 rpm. Bars indicate standard deviation.
Similar trends have been reported with C. rugosa lipase
covalently immobilized on poly(glycidylmethacrylate-
oleate, carried out in hexane at 60 ◦C, as reaction model. The influ-
ence of three main esterification parameters was tested.
methylmethacrylate) magnetic beads [49].
3.8.1. Effect of amount of lipase
3.7. Effect of solvent on lipase stability
The influence of enzyme amounts on the esterification reaction
of oleic acid and butanol was revealed in Fig. 5A. The percent-
age conversion increased with the increase in lipase amount and
remains constant with an enzyme load of 100 U (0.2 g). The excess
in enzyme did not contribute to the increase of the conversion yield.
At saturation point, all the substrates are bound to the enzyme and
added enzyme molecule could not find any substrate to serve as a
reactant. This was reported for the esterification of oleic acid cat-
alyzed by R. oryzae lipase [51]. According to Bloomer et al. [52] the
amount of enzyme would influence the total reaction time, which
is required to achieve desired conversion in esterification reaction.
The use of enzymes in organic medium is of great importance
as it allows the occurrence of esterification reactions which are
difficult to occur in presence of a high amount of water.
However, it is well known that enzyme activity could be strongly
affected by the organic solvent which may bring about the denatu-
ration of the enzyme thus leading to the loss of the catalytic activity.
The activity of the immobilized lipase was monitored in different
organic solvents varying from polar to non-polar. Among the six
tested solvents, immobilized TTL has a high tolerance for water-
immiscible organic solvents such as hexane, for which the activity is
maintained during long time incubation. Interestingly, the immobi-
lized TTL showed a high stability in the presence of water-miscible
organic solvents, since it retained almost 100% activity after expo-
sure, for 2 h at room temperature, to 40% of butanol, methanol,
ethanol, 2-propanol or acetone (Fig. 3). The loss of 25% of immobi-
lized TTL activity after its exposure to acetone for 120 min could be
due to the fact that this more polar solvent could strip off the essen-
tial water layer around the enzyme and thus distort the catalytic
conformation of the enzyme [50].
3.8.2. Effect of initial addition of water
Water plays an essential role in the lipase-catalyzed esteri-
fications. It is a well-known fact that water content affects the
equilibrium of esterification/hydrolysis reactions as well as the dis-
tribution of products in the media as the result of water acting as a
substrate [53]. Although the accurate amount of water for a given
enzymatic reaction depends on system components (enzyme, solid
support and solvent). Generally, it is reported that the optimum
level of added water is within the range of 0.2–3% based on dry
enzyme [54].
3.8. Esterification activity of the immobilized TTL
The effect of initial water content on enzymatic activity was
examined through the addition of water ranging from 0% to 10%
(w/w) of the total amount of reaction mixture. The synthesis in
The ability of the chitosan-immobilized TTL to catalyze the syn-
thesis of esters was investigated by taking the synthesis of butyl

I.B.-B. Romdhane et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 230–239
237
Fig. 6. GC/MS analyses of butyl ester obtained by esterification of oleic acid using T. thermophilus lipase. (A) Chromatographic profile of butyl oleate after 180 min of
esterification reaction. (B) Mass spectrum of butyl oleate compared to the GC/MS library.
hexane was carried out with 100 U (0.2 g) of immobilized lipase. As
shown in Fig. 5B, the conversion percentage was relatively higher
without initial added water. This may be due to the fact that water
already exist in substrates and enzyme preparation, as well as the
water produced during the esterification reaction was sufficient to
moist the enzyme and to render it active. The high hydrophilicity of
chitosan may explain why the conversion percentage is high with-
out addition of water. In fact, when water is added, the support
becomes more hydrated and so was the microenvironment of the
immobilized lipase. As a consequence, the esterification reaction
was thermodynamically disfavoured with respect to the hydrolysis
reaction.
3.8.3. Effect of molar ratio of substrates
The last important variables affecting the conversion percent-
age in esterification reactions is the relative proportions of the

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I.B.-B. Romdhane et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 230–239
substrates. The esterification was carried out with various molar
ratio oleic acid/butanol (1:1, 1:2, 1:3, 2:1 and 3:1) at the conditions
described above. Results depicted in Fig. 5C show that the high-
est conversion yield was obtained at a stoichiometric ratio close
to 1:1 between oleic acid and butanol after 180 min of reaction.
The conversion yield decrease gradually with the increase in acid
or alcohol. On the one hand, this observation may reflect the abil-
ity of the alcohol excess to distort the essential water layer from
lipase, which disrupts the conformation of the protein structure.
On the other hand, the presence of an excess of substrates can lead
to the competitive binding of either acid or alcohol to the immobi-
lized lipase due to the hydrophilic character of the chitosan matrix
that favours the accumulation of substrate molecules with their ter-
minal polar group pointed toward the surface. Thus, the excess of
one of the reagents will hinder the interaction frequency between
substrates and the immobilized lipase [55].
their kind in the SEM analysis. We also thank Miss L. Jlayel (CBS,
Sfax) for his help in the GC–MS analysis.
This work received financial support from “Ministère de
l’Enseignement Supérieur de la Recherche Sientifique et de la
Technologie, Tunisia” granted to the “Laboratoire de Génétique
Moléculaire des Eucaryotes” (Centre des Biotechnologies de Sfax,
Tunisia).
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