In situ immobilization of Candida antarctica B lipase in polyurethane foam support

Article abstract of DOI:10.1016/j.molcatb.2015.12.003

Candida antarctica B (CALB) lipase was immobilized using polyurethane (PU) foam as support by confinement method. This can be a promising technique due to the low cost of the support, simple procedure of immobilization and the possibility of using the derivative as catalyst for the food industry reactions. CALB immobilization on PU was performed using 6 mL polyol and 4 mL isocyanate (60-40%, v/v), with 1 mL of enzymatic solution (0.8 g enzyme in 5 mL distilled water). The immobilization process resulted in a 535% increase in activity for the enzymatic derivative. The enzyme was stable for 360 days at room (10-25 °C) and low (2-8 °C) temperatures, for 300 days at 40 and 60 °C and for 150 days at 80 °C in wet ambient conditions. The synthesis of geranyl propionate and ethyl oleate catalyzed by the immobilized derivative presented conversions of around 97 and 83%, respectively. The immobilized CALB in PU was reused consecutively different behavior occurred which stored in dry ambient conditions at different temperatures (room: 10-25 °C, refrigeration: 2-8 °C and 40 °C) resisted 30 cycles, for both forms of storage, keeping more than 87, 95 and 83% of its activity, respectively.

Full text of DOI:10.1016/j.molcatb.2015.12.003

Journal of Molecular Catalysis B: Enzymatic 124 (2016) 52–61
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
In situ immobilization of Candida antarctica B lipase in polyurethane
foam support
Nadia Ligianara D. Nyaria,∗, Ilizandra A. Fernandesa, Cindy E. Bustamante-Vargasa,
Clarisse Steffensa, Débora de Oliveirab, Jamile Zenia, Elisandra Rigoc,
Rogério M. Dallagoa,∗
a Department of Food Engineering, Regional and Integrate University of Upper Uruguay and Missions, URI - Erechim, Av. Sete de Setembro 1621, Erechim,
RS 99700-000, Brazil
b Department of Chemical and Food Engineering, Federal University of Santa Catarina, UFSC, Campus Universitário, Bairro Trindade, Caixa Postal 476,
Florianópolis, SC 88040-900, Brazil
c Department of Food Engineering, Federal University of Santa Catarina UFSC, PO Box 476, 88040-900 Florianópolis, SC, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Candida antarctica B (CALB) lipase was immobilized using polyurethane (PU) foam as support by confine-
ment method. This can be a promising technique due to the low cost of the support, simple procedure of
immobilization and the possibility of using the derivative as catalyst for the food industry reactions. CALB
immobilization on PU was performed using 6 mL polyol and 4 mL isocyanate (60–40%, v/v), with 1 mL of
enzymatic solution (0.8 g enzyme in 5 mL distilled water). The immobilization process resulted in a 535%
increase in activity for the enzymatic derivative. The enzyme was stable for 360 days at room (10–25 ◦C)
and low (2–8 ◦C) temperatures, for 300 days at 40 and 60 ◦C and for 150 days at 80 ◦C in wet ambient
conditions. The synthesis of geranyl propionate and ethyl oleate catalyzed by the immobilized derivative
presented conversions of around 97 and 83%, respectively. The immobilized CALB in PU was reused con-
secutively different behavior occurred which stored in dry ambient conditions at different temperatures
(room: 10–25 ◦C, refrigeration: 2–8 ◦C and 40 ◦C) resisted 30 cycles, for both forms of storage, keeping
more than 87, 95 and 83% of its activity, respectively.
Received 23 September 2015
Received in revised form 1 December 2015
Accepted 2 December 2015
Available online 7 December 2015
Keywords:
Immobilization
Lipase (CALB)
Polyurethane
Stability
Reuse
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
Thus, enzyme immobilization techniques appear as alternative
to solve the inconvenient mentioned above, allowing the enzyme
The enzymatic technology presents significant relevance since
represents promising tools for compounds synthesis of high com-
mercial value. Besides, enzymes show many advantages compared
to inorganic catalysts, as higher specificity, lower energy consump-
tion and higher catalytic activity [1,2].
Even with the benefit of this technology, replacement of chem-
ical catalysts by enzymes is not generalized in industrial processes
due to the instability, final product contamination by the catalyst
solution, difficulty of eliminating the catalyst in final product and
impossibility of recuperation and reutilization [3–5].
recovery and reuse, with displacement in pH value, adjustable
porosity, low toxicity, favorable mechanical properties, tempera-
ture and stability (thermal, operational and storage), allowing the
biotechnology process becomes economically viable [3–11].
The techniques usually employed to immobilize enzymes on
solid supports are based on physical and chemical mechanisms,
global activity parameters of the immobilized, regeneration and
deactivation characteristics, procedure cost and low toxicity of
reagents [7,12,13]. The enzyme immobilization can occur by
adsorption or binding in an insoluble material or cross-linking and
encapsulation in inorganic/organic polymer matrices that provides
new possibilities for use in materials science [12,14–17].
In this context, new immobilization processes and industrial
applications have been related in the literature, principally in syn-
thetic processes that possess a broad range of catalytic activities
suitable for biotransformation [1,2,12], food and flavor produc-
∗
Corresponding authors.
E-mail addresses: nadialigianara@hotmail.com (N.L.D. Nyari),
dallago@uricer.edu.br (R.M. Dallago).
http://dx.doi.org/10.1016/j.molcatb.2015.12.003
1381-1177/© 2015 Elsevier B.V. All rights reserved.

N.L.D. Nyari et al. / Journal of Molecular Catalysis B: Enzymatic 124 (2016) 52–61
2. Material and methods
53
2.1. Materials
The enzyme used was lipase from CALB (Novozyme NZL-102-
LYO-HQ) obtained in lyophilized form, from Novozymes Latin
America LTDA. The commercial polyol and isocyanate monomers
used in this work were produced for a specific formulation
for mattresses and foam injected by the Company Flexible
Polyurethanes–Mannes (Erechim, RS, BR).
2.2. Enzyme and protein profiles of commercial lipase
The protein content of the lipase was determined by Bradford
[24], using serum bovine albumin as standard. The characteriza-
tion of the enzyme profile was carried out by determination of
CALB activity. The specific activity (U mg−1) was determined by
the quotient of enzyme activity (U mL−1) and total protein content
(mg mL−1).
2.3. Immobilization of CALB lipase in polyurethane (PU)
CALB immobilization on PU was performed using 6 mL polyol
and 4 mL isocyanate (60–40%, v/v), with 1 mL of enzymatic solution
(0.8 g enzyme in 5 mL distilled water). The enzymatic solution was
added in the polyol and homogenized. After, isocyanate was added
at constant stirring. The in situ polymerization was conducted at
20 ◦C, until growing the PU foam (5 min), remaining stationary for
3 h and then crushed, producing a homogeneous product. 1 g of this
product was washed with 5 mL of buffer solution (sodium phos-
phate pH 7). It was evaluated the enzyme leaching from the support
by the measurement of esterification activity of the wash solution.
2.4. Characterization of immobilized CALB lipase
2.4.1. Apparent density
The apparent density was determinate according to the method-
ology described by American Society for Testing and Materials
(ASTM) in Standard ASTM D 3574-03. The measurement of sam-
ples was performed with a caliper. The PU foam was cut into
2 × 2 × 2 mm2. Then, it was weighed in a digital scale with a preci-
sion of 0.0001 g (Shimadzu). The value calculated was expressed in
g/cm3. Analyses were performed in triplicate.
Fig. 1. SEM microscopy of polyurethane foam used as support without CALB lipase
(a) and (b) CALB immobilized in situ polyurethane.
tion, pharmaceuticals, carbohydrate esters synthesis, amines and
amides, cosmetics, among others [1,12,18].
2.4.2. Morphology characterization using scanning electron
microscope (SEM)
Within the immobilization process, it can be cited the method-
ology developed by our research group, where PU was used as
support with aggregated enzyme, in situ, at one monomer, permit-
ting the enzyme stays entrapped on the impermeable polymeric
matrices. This simple process allow the free diffusion of substrate
and reaction products, not been enfold by physical, chemical or
covalent bindings, between the enzyme and support [19,20].
In this way, keeping their mobility, reducing enzyme loss and
controlling pore size can be possible application at a large scale
with low cost. Also present easy interaction between enzyme and
support in organic medium, simple separation reaction product,
minimizing the enzyme loss and contamination [1,10–12,21–23].
Also, it is important to emphasize that the reaction time required
in this immobilization process is low (about 5 min) to the total
incorporation of the offered enzyme and uniform distribution in
the support [20]. Thus, this work aimed expands the application
of lipases immobilization methodology. Candida antarctica (CALB)
lipase was used to monitoring the distribution in PU support. Stabil-
ity at different temperatures, pH, recycles and potential application
in esters synthesis were also evaluated.
Morphology of PU support and immobilized CALB lipase in PU
support were evaluated by scanning electron microscope (SEM)
(DSM960 Zeiss). The samples were mounted on conductive carbon
tape (double-sided) adhered to an aluminum sample port and sput-
ter coated with gold in a sputtering unit (SCD050/LEICA) at 40 mA
for 90 s. Next, were characterized with SEM operating at 10 kV.
2.4.3. X-ray diffraction (XRD)
X-ray diffractograms (XRD) were collected in a X-ray diffrac-
tometer (Rigaku, Miniflex II) with a CuK␣ radiation source. Samples
of immobilized CALB lipase in PU support were scanned. Scans were
executed from 5◦ to 35◦ (2 h) at a speed of 2 min−1
.
2.5. Immobilization yield
The immobilization yield was calculated considering the total
activity of the free enzyme in solution (volume of the enzyme
extract used in the immobilization experiment). The total activity

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N.L.D. Nyari et al. / Journal of Molecular Catalysis B: Enzymatic 124 (2016) 52–61
Fig. 2. X-ray diffractograms of the immobilized CALB on polyurethane support.
of the immobilized enzyme (total mass of the immobilized product)
was calculated according to Eq. (1).
ated by the measurement of the esterification activity of enzymatic
derivative using oleic acid and ethanol as substrates.
The reuse process was evaluated in two ways, one with
sequential recycles with and without washing with hexane
(Sigma–Aldrich) and other with sequential recycles, for 24 h of
drawing and washed singly with ethanol, acetone (Sigma–Aldrich)
and buffer solution (sodium phosphate pH 7). For the sequen-
tial recycles study without hexane washing, it was performed a
reaction (ethanol and oleic acid) with the immobilized deriva-
tive and measured the esterification activity. Next, the excess of
reaction medium was removed (4 mL) and added 5 mL of new reac-
tion medium, and this process was repeated until loss of 50% of
initial activity of the immobilized derivative. Then, it was cen-
trifuged for 4 min at 2000 rpm, filtered and placed in an oven
at 40 ◦C for 30 min to evaporate the solvent. After, 5 mL of the
new reaction medium was added, and this process was repeated
also until loss of 50% of initial activity of the immobilized deriva-
tive. The sequential recycle process of 24 h occurred with three
reactions (ethanol and oleic acid) with the immobilized deriva-
tive followed by the measurement of the esterification activity.
Later, the excess of reaction medium was removed (4 mL) and
washed singly with 4 mL of ethanol, acetone and buffer solu-
tion (sodium phosphate pH 7). This was centrifuged for 4 min
at 2000 rpm, filtered and placed in an oven at 40 ◦C for 24 h to
evaporate the solvent. After, 5 mL of the new reaction medium
was added, and this process was repeated until loss of 50%
of initial activity of the immobilized enzyme derivative (Eq.
(2)).
UTEXP
UT0
RI % =
( )
× 100
(1)
where RI (%): immobilization yield; UTExp: is the total activity of
the immobilized enzyme; UT0: total activity of the enzyme solution
offered for immobilization.
2.6. Partial characterization of the immobilized enzyme
The thermal stability of the free and immobilized CALB was
determined at room temperature (10–25 ◦C), refrigeration temper-
ature (2–8 ◦C) and 40–160 ◦C. The effect of pH on immobilized lipase
activity in PU was also evaluated. CALB was incubated at 10–20 ◦C,
with the pH varying from 3 to10. The residual activity was used to
evaluate the storage activity (thermal stability), calculated using as
reference the initial activity for each system, according to Eq. (2).
UX
AR % =
( )
× 100
(2)
Uinitial
where AR (%): residual activity; Uinitial and UX are the activities at
t beginning and after incubation at evaluated temperature, respec-
tively.
2.7. Immobilized CALB lipase operational evaluation
The greatest interest in immobilizing enzymes is to promote
reuse. In this way, the recyclability of immobilized CALB was evalu-

N.L.D. Nyari et al. / Journal of Molecular Catalysis B: Enzymatic 124 (2016) 52–61
55
tion of free (0.2 g enzyme solution—0.8 g enzyme in 5 mL of distilled
water) and immobilized CALB (0.1 g immobilized solution).
The amount of acid consumed was determined by titration with
0.05 M NaOH. One unit of enzyme activity was defined as the
amount of enzyme which consumes 1 ␮mol of fatty acid per minute
under the assay conditions. The enzyme activity was calculated
using Eq. (3).
(Vb − Va) × M × 1000 × Vf
EA =
(3)
t × MEL × VC
where EA: esterification activity (U/g); Va: NaOH volume spent in
the sample titration withdrawn after 40 min (mL); Vb: NaOH vol-
ume spent in the sample titration withdrawn after 0 min (mL); M:
NaOH solution molarity; Vf: final volume of reaction medium (mL);
t: time (min); MEL: weight of free enzyme (enzymatic solution) or
immobilized; Vc: aliquot volume of reaction medium removed for
titration (mL).
2.9. Application of immobilized CALB lipase in catalytic synthesis
reaction
2.9.1. Ethyl oleate
The evaluation of the ability of immobilized CALB to catalyze the
ethyl oleate synthesis was performed using oleic acid and ethanol
as substrates, at different molar ratios (1:1, 1:3 and 3:1) (5 g of sub-
strates), immobilized CALB 0.1 g, temperature of 40 ◦C, 160 rpm for
40 min, conditions described before (item 2.8). Then, 500 ␮L were
removed (in triplicate) from the reaction medium and placed in
15 mL of acetone–ethanol solution (1:1) (v/v).
The enzymatic activity determination was performed in tripli-
cate. The amount of acid consumed was determined by titration
with 0.05 M NaOH until pH 11, using (Eq. (3)). When the acid is
in excess in the reaction, the volume of alcohol is the limiting,
molar ratio acid/ethanol (3:1), using Eq. (4). With 100% conversion,
the maximum consumption of acid in excess, results in 1/3 that is
offered in the reaction.
Vb NaOH − VR NaOH
(
)
(
)
EC =
× 100
(4)
Vb NaOH
(
)
where EC: esters conversion (%); Vb: volume of NaOH used in the
titration of white (mL); VR: volume of sample (acid) consumed after
the reaction (mL); 1/3 Vb: NaOH volume of white consumed in the
titration (mL) (acid in excess).
Determination of reaction conversion was then also accom-
plished by computing the difference between initial (original) and
final (after the reaction) acidity of the solution Eq. (5) [27].
C0 − C
%Molar =
× 100
(5)
C0
where C0: denotes the acidity of the original solution and C: the
acidity of the solution leaving the process.
2.9.2. Geranyl oleate
CALB immobilized derivative was evaluated as catalysts in
esterification, methodology described by Paroul et al. [25,26] and
Trentin et al. [27] which used geraniol (Safc, 99% purity), oleic acid
(Sigma–Aldrich, 99% purity), as the substrates in different molar
ratios (1:1, 1:3 and 3:1) (5 g of substrates), 0.1 g immobilized CALB,
temperature of 40 ◦C, 160 rpm for 6 h. The quantification by titra-
tion with sodium hydroxide (0.05 M NaOH, Vetec) until pH 11 was
carried out and the product quantification was by Eqs. (3)–(5).
Fig. 3. Free CALB lipase stability at different temperatures (room 10–25 ◦C and cool-
ing 2–8 ◦C) (a), temperatures at (40, 60 and 80 ◦C) (b) and temperatures at (100, 120,
140 and 160 ◦C) (c).
2.8. Enzyme activity
The esterification reactions were performed by preparing a reac-
tion mixture of oleic acid and ethanol (1:1 molar ratio) as the
reactants, according the methodology described by Paroul et al.
[25,26]. The activity measurement was carried out with the addi-
2.9.3. Geranyl propionate
The enzymatic synthesis was performed according the method-
ology described by Paroul et al. [25,26], which used geraniol and
acid propionic (Merck, 99% purity) as the substrates in different

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2.9.4. Statistical analysis
Each experiment was done in triplicate. Data were expressed
as means standard deviation, and subjected to one-way analy-
sis of variance (Tukey) using Statistica 8.0 (StatSoft) software. A
significance level of 95% (p < 0.05) was used.
3. Results and discussion
3.1. Immobilization of CALB lipase in polyurethane (PU)
3.1.1. Immobilization yield
The immobilization yield was defined as the relationship
between the total activity offered (UT0), calculated considering
the amount of free enzyme (0.2 g) used in the incorporation step,
and the experimental total activity (UTExp.), calculated by taking
account the total activity of the PU support incorporated with the
enzyme (10.8 g). The UT0 and UTExp values obtained for the free and
immobilized enzymes, respectively, are shown in Table 1.
There was an increase in the activity of the immobilized enzyme
(up 535.3%) in relation to the free enzyme. These results suggest
a beneficial effect of immobilization in the enzyme activity. This
trend may be associated with several factors, such as the easy acces-
sibility of new active sites. In order to evaluate the efficiency of
the immobilization process (enzyme on PU support), the immo-
bilized derivative was submitted to washing with buffer solution
(sodium phosphate pH 7). It was verified that the washing solution
no showed esterification activity, indicating that all enzyme added
to the support was irreversibly incorporated by confined method
and/or covalently bound to PU.
Ferreira-Dias et al. [28] define that during the process of poly-
merization (polyurethane) the enzyme becomes entrapped in the
foam matrix, covalent binding between carboxylic acid and amina
groups of the lipase and free TDI + MDI (support) radicals of the
prepolymer may also occur. Forde et al. [29], CALB has a preference
for carriers with larger pore diameter allowing for easier access
for the enzyme and more space for the substrate to entry and for
the reaction to occur [30]. If the pore diameter of the support is
equal or slightly bigger than the enzyme size (diameter), it will be
difficult or even impossible the immobilization inside the support
particle. The results obtained in the current work are superior to
those in previous reports in the literature by Cipolatti et al. [31],
Bustamante-Vargas [32], Silva [33] and other.
3.1.2. Characterization of immobilized CALB lipase
The apparent density is the ratio between the geometric volume
and specimen mass, including the polymeric material and the gas
inside the cells in the PU foam. The
density values were calculated for all immobilized deriva-
tives, showing values of 0.029 0.015 g cm−3. Since this measure
considers the reaction process of expanding foam and mass,
it is assume that the foam expansion process occur uniformly
(0.627 0.140 g cm−3) [34].
With the use of SEM images, it is possible to verify that the
pores formed during synthesis are homogeneous or not, and also to
observe possible changes in the surface of the polymer structure.
SEM microscopy of the generated foam used as support for CALB
immobilization and immobilized derivatives are shown in Fig. 1a.
The empty spaces resulted from gas bubbles (CO2) released dur-
ing the PU reaction. The support morphology changed with the
presence of the CALB enzyme (Fig. 1b).
The X-ray diffractogram of immobilized CALB on PU (Fig. 2)
shows a broad peak, characteristic of amorphous material.
Similar behavior was verified by Silva et al. [33], where was char-
acterized pure PU and PU with clay and Fiorio [35] characterized
Fig. 4. Immobilized CALB lipase stability different temperatures (room 10–25 ◦C and
cooling 2–8 ◦C) (a), temperatures at (40, 60 and 80 ◦C) (b) and temperatures at (100,
120, 140 and 160 ◦C) (c).
molar ratios (1:1, 1:3 and 3:1) (5 g of substrates). The quantifica-
tion of geranyl propionate was carried out by titration with sodium
hydroxide (0.05 M NaOH) until pH 9.1, using Eq. (3). When the acid
is in excess in the reaction, the volume of alcohol is the limiting,
molar ratio acid/ethanol (3:1), using Eq. (4). With 100% conversion,
the maximum consumption of acid in excess, results in 1/3 that is
offered in the reaction (Eq. (5)).

N.L.D. Nyari et al. / Journal of Molecular Catalysis B: Enzymatic 124 (2016) 52–61
57
Fig. 5. Immobilized CALB lipase stability immersed in reaction medium submitted to high temperatures (40, 60 and 80 ◦C).
Fig. 6. Immobilized CALB lipase stability immersed in reaction medium submitted at different pH (3–10).

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Table 1
Effect of the initial amount of CALB lipase on the enzyme activity (U g−1) and yield of immobilization (%).
CALB free
CALB immobilized in PU
Specific activity
Enzyme mass
(g)
Activity
(U g−1
Offered activity
(UTo
Specific activity
PU + CALB
Mass
immobilized (g)
Total activity
(UTExp
Yield
(%)
(U/mg−1
)
)
)
(U/mg−1
)
activity (U g−1
)
)
1753.24
0.2
3057.5
611.5
1002.98
302.9
10.8
3271.5
5535.1
Fig. 7. Behavior of immobilized CALB lipase without and with hexane washes in continuous cycle.
the same support and observed the presence of a broad peak in
absorbs a lot of energy, it may undergo conformational changes at
the active sites, which are sometimes irreversible and may even
completely denature the enzyme [36].
2ꢀ–20◦.
3.2. Free and immobilized enzyme stability at different
temperatures
3.2.2. Immobilized enzyme
The determination of immobilized enzyme stability at differ-
ent temperatures was performed by two ways: storing the dry
immobilized enzyme at different temperatures, and storing the
immobilized enzyme in the reaction medium (oleic acid and
ethanol as the active principal components).
3.2.1. Free enzyme
Regardless of the process conditions, the thermal stability is
often a key factor in the success of bioprocesses, since the reac-
tion rates typically increase exponentially with the temperature
until the point of denaturation of the enzyme. The results of the
free enzyme stability at room temperature (10–25 ◦C) and refriger-
ation temperature (2–8 ◦C), shown in Fig. 3a, demonstrates enzyme
inactivation over 40 days (960 h) of storage under these conditions.
Fig. 3b shows that the enzyme activity was retained at 40% for
24 h at 40 ◦C and 60 ◦C. At 80 ◦C the enzyme lost 83% of its initial
activity after 5 h. However, when the enzyme was stored at 120,
140 or 160 ◦C, complete inactivation was observed within 45 min
(Fig. 3c). At 100 ◦C, the enzyme maintained 30% of its residual activ-
ity after 60 min.
It was possible to observe that the immobilized enzyme stored
under dry ambient conditions at room temperature (10–25 ◦C) and
refrigeration temperature (2–8 ◦C), remained stable during 360
days of storage, showing a residual activity of 87% when stored
at 10–25 ◦C and 51% at 2–8 ◦C (Fig. 4a). These results not represent
great losses in residual activity, indicating that the immobilized
enzyme was more efficient than the free enzyme under the same
conditions. Fig. 4b shows the behavior of the immobilized enzyme
submitted at 40, 60 and 80 ◦C. The immobilized enzyme presented
residual activity of 60 and 57% after 300 days of storage at 40
and 60 ◦C, respectively, and 41% at 80 ◦C after 150 days. These
results demonstrate an improvement in the stability compared to
the free enzyme, which presented total inactivation in less than
24 h under these conditions. Under extreme temperature condi-
tions, the immobilized enzyme showed residual activity of about
Nicoletti et al. [34] studied the behavior of free CALB lipase and
noted that under different temperatures (25–80 ◦C) remained sta-
ble over 48 h. Soluble enzymes are complex protein molecules with
a certain degree of flexibility; catalytic activity requires a highly
ordered structure, which is very delicate and fragile. If the molecule

N.L.D. Nyari et al. / Journal of Molecular Catalysis B: Enzymatic 124 (2016) 52–61
59
Fig. 8. Behavior of the immobilized CALB lipase at different temperatures (room: 10–25 ◦C, cooling: 2–8 ◦C and 40 ◦C) when stored after drying (a) and when stored in reaction
medium (b).
40% at 100 ◦C after 5 days, 50% at 120 ◦C after 10 days, 56% at 140 ◦C
after 21 days and 40% at 160 ◦C after 20 days (Fig. 4c).
Thus, evidencing good resistance to elevated temperatures, the
results demonstrate the effectiveness of the catalyst in PU matrix.
Also, the catalyst may have been more stable, or modifications in
the support structure may have favored immobilized enzyme sta-
bility [37]. The enzyme in soluble form has some flexibility, which
causes conformational changes in active site, often irreversible, and
makes it inactive for direct temperature influences. When immo-
bilized, it become more rigid due to their bonds with the support,
decreasing the flexibility while maintaining the shape of the active
site, which is responsible for its activity.
The PU support has great mechanical stability, this contributed
to present a higher thermal stability. In this way, exposure of the

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N.L.D. Nyari et al. / Journal of Molecular Catalysis B: Enzymatic 124 (2016) 52–61
Table 2
immobilized enzyme to the reaction medium, in this case, favored
the increased stability of immobilized CALB on PU.
Reaction yield obtained in the catalyzed reactions by immobilized CALB lipase in
polyurethane.
It is therefore purposed that this immobilized enzyme can be
used in future applications employing a continuous flow rate at
different temperatures. The results obtained in the current work
are superior to those in previous reports in the literature by
Bustamante-Vargas et al. [32] and Ondul et al. [38].
When the immobilized enzyme was stored in the reaction
medium (oleic acid and ethanol) for 150 days, the enzyme remained
80, 71 and 63% of the original activity at 40, 60 and 80 ◦C, respec-
tively (Fig. 5).
In this way, exposure of the immobilized enzyme to the reaction
medium, in this case, favored the increased stability of immobi-
lized CALB on PU. It is therefore purposed that this immobilized
enzyme can be used in future applications employing a continuous
flow rate at different temperatures. The results of this study are
better than those reported by Guncheva et al. [39], Hernandez and
Fernandez-Lafuente and [44] Fernandes et al. [40] these authors
also highlighted that this process could preserve enzyme activity
at higher temperatures, and was even twice as active as the native
enzyme.
Enzymatic synthesis
Yield (%)
Molar ratio (acid/alcohol)
(1/3)
(3/1)
(1/1)
Geranyl oleate
Geranyl propionate
Ethyl oleate
10.3
96.6
96.6
26.5
97.3
98.0
33.9
97.7
99.9
3.5. Application of immobilized CALB in catalytic syntheses
reactions
The immobilized CALB was used in esterification reactions,
since it presented high activity, stability and recyclability in these
reactions. The results of conversions obtained in the enzymatic syn-
thesis reactions of geranyl oleate, geranyl propionate and ethyl
oleate esters. The enzymatic synthesis of geranyl oleate, geranyl
propionate and ethyl oleate production was carried out at differ-
ent molar ratios (acid/alcohol (1:1, 1:3 and 3:1)). The conversion
of geranyl oleate, at a molar ratio of 1:1, was 33.9%. Geranyl pro-
pionate presented conversion of around 97%, at all molar ratios,
and the same behavior was observed for ethyl oleate with 83.2%
conversion (Table 2).
3.3. Effect of pH on the immobilized lipase activity
The results of the current study demonstrate the potential appli-
cation of this immobilized enzyme in synthesis reactions, especially
for geranyl propionate and ethyl oleate. Similar results were found
by Paroul et al. [25,26] and Nicoletti et al. [34]. Considering the
high conversion and low cost of citronella oil, it can be confirmed
by results obtained in this work that the substitution of commer-
cial alcohols by essential oils can contribute to make the process
economically viable.
Immobilized CALB was stored at different pH levels (3–10) at
ambient temperature (10–25 ◦C) to evaluate the changes in lipase
interaction with the environment. The residual activity was mea-
sured after 30 days, showing 68, 68, 100, 88, 75, 55, 59 and 74%
residual activity at pH 3, 4, 5, 6, 7, 8, 9 and 10, respectively (Fig. 6).
Dhake et al. [41] showed that a change in pH had no significant
effect on immobilized enzyme activity, with the optimum pH in
the range of 5–8. It was also shown that the immobilized enzyme
immersed in neutral and basic solutions had greater activity. It has
been well documented that the optimum pH range of yeast lipases
is generally between pH 5 and 8, with a few exceptions of lower pH
optima of 2.0 [42–44].
4. Conclusions
The immobilization process resulted in a 535% increase in
enzyme activity compared to the free enzyme. This result sug-
gests a beneficial effect of immobilization on enzyme activity. The
immobilized enzyme application in geranyl propionate and ethyl
oleate synthesis resulted in 97 and 83% conversion, respectively.
This study indicates that immobilization CALB is a promising tech-
nique, especially given the simplicity of the process, its low cost
and the ability of supported applications in biocatalysis reactions
of interest to the food industry.
The results obtained in the current work are superior to those
in previous reports in the literature by Liu et al. [45].
3.4. Evaluation of the recyclability of the immobilized lipase
Another important parameter in enzyme-catalyzed reactions is
the evaluation of the biocatalyst reuse, in order to decrease the
cost of the process. Fig. 7 shows the behavior of the immobilized
CALB submitted to washing with and without hexane in a continu-
ous cycle. The results obtained after continuous recycling, with and
without hexane washes, presented distinct behavior, since after six
cycles, the immobilized enzyme washed with hexane had 49% of
its initial activity. On the other hand, a completely different behav-
ior occurred with the immobilized enzyme without washes, which
resisted 36 continuous cycles with 51% of the initial activity. This
fact can be associated with the non capacity of hexane modifying
the active site of the enzyme, leading to an increase in enzyme
activity, as already reported by [46].
Fig. 8a shows the behavior of the immobilized enzyme stored in
dry ambient conditions at different temperatures (room: 10–25 ◦C,
refrigeration: 2–8 ◦C and 40 ◦C) after each cycle of use Fig. 8b.
The immobilized enzyme stored at these temperatures resisted 30
cycles, for both forms of storage, maintaining more than 87, 95
and 83% of the activity at room temperature, refrigeration temper-
ature and 40 ◦C, respectively. The results obtained in the current
work are superior to those in previous reports in the literature by
Bustamante-Vargas et al. [32], Cadena et al. [47], Guncheva et al.
[48] and Barbosa et al. [49].
Acknowledgments
The authors would like to thank URI-Campus de Erechim, CNPq,
CAPES and FAPERGS for the infra-structure and financial support
for this research.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.molcatb.2015.12.
003.
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