Covalent immobilization of lipase onto amine functionalized polypropylene membrane and its application in green apple flavor (ethyl valerate) synthesis

Article abstract of DOI:10.1016/j.procbio.2010.09.014

In this study, a functionalized hydrophobic polypropylene chloride membrane (PPC) was prepared by the amination of chlorinated polypropylene with hexamethylene diamine (APP). The PPC and APP membranes were characterized using SEM, FTIR and contact angle studies. The aminated polypropylene (APP) membrane was used for covalent immobilization of Candida rugosa lipase via glutaraldehyde coupling. The retained activity of the immobilized lipase was 76%. Kinetic analysis shows that the dependence of lipolytic activity of both free and immobilized lipase on tributyrin substrate concentration can be described by Michaelis-Menten model. The estimated apparent K_m values for the free and immobilized lipase were 2.9 and 8.4 mM, respectively. The V_max values of free and immobilized enzymes were calculated as 926 and 741 U/mg enzyme, respectively. Optimal temperature was 5 °C higher for immobilized enzyme than that of the free enzyme. Thermal and storage stabilities were found to be increased upon immobilization. Finally, the immobilized lipase was used for the production of green apple flavor (i.e., ethyl valerate) in hexane medium.

Full text of DOI:10.1016/j.procbio.2010.09.014

Process Biochemistry 46 (2011) 372–378
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Covalent immobilization of lipase onto amine functionalized polypropylene
membrane and its application in green apple flavor (ethyl valerate) synthesis
a,∗
b
a
a
Gülay Bayramo g˘ lu , Baki Hazer , Begum Altınta s¸ , M. Yakup Arıca
a
Biochemical Processing and Biomaterial Research Laboratory, Faculty of Arts and Sciences, Gazi University, 06500 Teknikokullar, Ankara, Turkey
Department of Chemistry, Zonguldak Karaelmas University, 67100 Zonguldak, Turkey
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 June 2010
Received in revised form
In this study, a functionalized hydrophobic polypropylene chloride membrane (PPC) was prepared by
the amination of chlorinated polypropylene with hexamethylene diamine (APP). The PPC and APP mem-
branes were characterized using SEM, FTIR and contact angle studies. The aminated polypropylene (APP)
membrane was used for covalent immobilization of Candida rugosa lipase via glutaraldehyde coupling.
The retained activity of the immobilized lipase was 76%. Kinetic analysis shows that the dependence
of lipolytic activity of both free and immobilized lipase on tributyrin substrate concentration can be
described by Michaelis–Menten model. The estimated apparent Km values for the free and immobilized
lipase were 2.9 and 8.4 mM, respectively. The Vmax values of free and immobilized enzymes were calcu-
1
8 September 2010
Accepted 20 September 2010
Keywords:
Polypropylene membrane
Immobilized enzyme
Enzyme stability
Enzyme kinetics
Lipase
◦
lated as 926 and 741 U/mg enzyme, respectively. Optimal temperature was 5 C higher for immobilized
enzyme than that of the free enzyme. Thermal and storage stabilities were found to be increased upon
immobilization. Finally, the immobilized lipase was used for the production of green apple flavor (i.e.,
ethyl valerate) in hexane medium.
Ethyl valerate
©
2010 Elsevier Ltd. All rights reserved.
1
. Introduction
cal reaction conditions. Hydrophilic groups could be introduced
on the PP structure by covalent attachment of functional –NH2
group [17,18]. Hydrophilic functional groups containing hydropho-
bic support could be obtained and would be used in enzyme
immobilization studies. When considering inherent properties of
lipases, they are activated on interface, and hence, the immobiliza-
tion of lipases on the hydrophobic membrane surface seems to be
the best solution [9,12]. In a previous study, polypropylene micro-
filtration membrane was modified by grafting with hydrophilic
hydrophobic groups containing polymers. Lipases from Candida
rugosa were immobilized via adsorption on these modified mem-
branes surface [6].
Lipases (E.C. 3.1.1.3) from different sources are currently used in
various biochemical reactions including triacylglycerols hydrolysis,
esterification between fatty acids and alcohols. The rising interest
in lipase mainly lies on its wide industrial applications, including
detergent formulation, oils/fats degradation, pharmaceuticals syn-
thesis, and cosmetics production [19–21]. There are several studies
about ester synthesis in hexane medium using immobilized lipase.
Ethyl valerate is one of the most important ester compound (green
apple flavor) used in the cosmetic and food industries [22,23].
In the present study, amino functionalized non-porous
polypropylene membrane was prepared using PPC and hexamethy-
lene diamine. The membrane matrix was used for the covalent
immobilization of Candida rugosa lipase after activation of glu-
taraldehyde. The influence of several parameters was characterized
such as activity retention, as well as kinetic properties, thermal and
Immobilization confers additional stability to a variety of
enzymes against several forms of denaturation. Enzymes have been
immobilized on different shapes of supports (i.e., membranes or
beads) either by covalent binding, entrapment or adsorption [1–4].
Enzyme immobilized on membrane may find practical application
in biosensors and enzyme reactors as less expensive, more stable
and reusable alternatives to free enzymes [5,6]. A variety of support
materials have been used for lipase immobilization for example
silica [7] magnetic poly(GMA-MMA) beads [8], polypropylene hol-
low fiber membrane modified with hydrophobic polypeptide [9],
polysulfone ion exchange membranes [10], mesoporous silica [11],
poly(hydroxyethyl methacrylate)-based membranes [12], calcium
alginate gels [13] and recently developed nano-structured mem-
brane matrices [14]. After immobilization of lipase, changes were
observed in enzyme activity, optimum pH, affinity to substrate and
stability. The extent of these changes depended on the source of
enzyme, the type of support and the method of immobilization. It
is, thus, important that the choice of proper support material and
immobilization method over the free bioactive agent should be well
justified [1,15,16]. Polypropylene (PP) is a hydrophobic polymer.
It is chemically inert and stable to many chemical and biologi-
∗ Corresponding author. Tel.: +90 312 202 1533; fax: +90 312 212 2279.
E-mail address: g bayramoglu@hotmail.com (G. Bayramo g˘ lu).
1
359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2010.09.014

G. Bayramo g˘ lu et al. / Process Biochemistry 46 (2011) 372–378
373
operational stability aspects were investigated and compared with
free enzyme. Finally, immobilized lipase was used for ethyl valerate
synthesis in both solvent free and hexane medium.
medium. After this period, the enzyme-immobilized APP membrane was imme-
diately transferred to ethylene diamine solution (5.0 mg/ml ethylene diamine) in
same buffer solution to block the free reactive groups on the membranes. Schematic
representation of chemical route for the enzyme immobilization is presented in
Fig. 1. Physically bound enzyme from the membrane was removed by washing
with a solution containing ionic detergent (sodium cholate, 50 mM) and salt (NaCl,
2. Materials and methods
1
.0 M) at pH 8.0 for 2 h. The amount of immobilized lipase on the APP membrane
2
.1. Materials
was determined by measuring the initial and final concentrations of protein within
the immobilization medium and washed solutions using Coomassie Brilliant Blue
Lipase (from Candida rugosa, Type VII, ≥700 units/mg solid, (specific activity:
[
(
24]. A calibration curve constructed with lipase solution of known concentration
0.05–0.50 mg/mL) was used in the calculation of protein in the enzyme and in wash
9
17 units/mg protein. The specific activity was based on protein content of the solid
and the protein content was determined by the Bradford method) and polypropy-
lene chloride (PPC; MW 150,000, chlorinated about each three repeating unit) were
supplied from the Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Hexamethy-
lene diamine (HMDA), valeric acid (pentanoic acid), and n-hexane were obtained
from Fluka AG (Switzerland). Glutaraldehyde, toluene, anhydride magnesium sul-
fate, tributyrin, gum Arabic and sodium cholate were obtained from Sigma Chem. Co.
solutions.
2
.4. Activity assays of lipase
The activity of free and immobilized lipase was determined by olive oil hydrol-
ysis as described previously [12]. A 100 ml olive oil emulsion was prepared by
mixing olive oil (50 mL) and gum Arabic solution (50 ml, 7%, w/v). The assay mix-
ture consisted of emulsion (5 mL), phosphate buffer (2.0 mL, 100 mM, pH 7.0) and
free enzyme (0.5 mL, 1.0 mg/mL) or immobilized enzyme (10 cm membrane). Oil
hydrolysis was carried out at 35 C for 30 min in a shaking water-bath at 150 rpm.
(
St. Louis, MO, USA). All other chemicals were of analytical grade and were purchased
from Merck AG (Darmstadt, Germany).
2
2.2. Preparation of polypropylene membrane for enzyme immobilization
◦
The reaction was stopped by the addition of 10 ml of acetone–ethanol solution (1:1,
v/v). The liberated fatty acid in the medium was determined by titration with 50 mM
NaOH solution. These activity assays were carried out over the pH range 4.0–9.0 and
temperature range 20–60 C to determine the pH and temperature profiles for the
free and the immobilized enzymes.
The preparation of aminated polypropylene membrane was carried out as
reported by Hong et al. [18]. Polypropylene chloride (3 g, MW 150,000) was dissolved
in a flask containing toluene (40 mL). Hexamethylene diamine (1.0 g) was dissolved
in acetone–isopropyl alcohol mixture (1:1 ratio (v/v), 50 mL). After dissolving each
chemical, these solutions were dried with anhydride magnesium sulfate, and then
were mixed in a round bottom two neck flask. It was equipped with a coiled con-
densing tube and with an argon gas inlet adaptor. The flask was transferred in a
◦
2
.5. Determination of the kinetic parameters enzyme preparation
◦
water-bath and it was stirred magnetically at 50 C for 48 h. Argon atmosphere was
The kinetic constants were determined using tributyrin as substrate in the con-
maintained during the reaction period. After reaction, the amino group function-
alized membrane was obtained by the casting of aminated polymer solution on a
Teflon plate. The membranes were dried at room temperature for 24 h and were
peeled from Teflon plate. They were washed several times with purified water, and
centrations range (5–50 mM) using free and immobilized lipase and titrating the
butyric acids produced with 50 mM NaOH as described above. The experiments
were conducted under the optimized assay conditions. The apparent Km and Vmax
values for the free and immobilized lipase were calculated from Lineweaver–Burk
plots by using the initial rate of the enzymatic reaction.
◦
◦
then were dried sequentially at 60 C and 105 C for 24 h and 12 h, respectively.
The activation of amino groups of the APP membranes was achieved by reaction
with glutaraldehyde. APP membranes (about 2 g) were transferred into glutaralde-
hyde solution (50 ml) and the concentration of glutaraldehyde was varied in the
range of 0.2–2.0% (v/v). The pH of the solution was adjusted to 7.0 with 0.1 M NaOH.
2.6. Thermal stability of the immobilized lipase
◦
After closing flask, the reaction was carried out at 25 C for 12 h, while continuously
The thermal stabilities of the free and immobilized lipase were determined by
incubation in substrate-free phosphate buffer solution (0.1 M, pH 7.0) at two differ-
stirring the reaction medium. At the end of this period, the activated membranes
were removed and washed several times with methanol and then dried in a vacuum
◦
◦
ent temperatures (55 C and 65 C) under continuous shaking at 150 rpm. At 15 min
time intervals, the remaining activities of the free and immobilized lipase were
measured as described above.
◦
oven for 6 h. They were then stored at 4 C until use.
2.3. Immobilization of lipase on APP membranes
2
.7. Operational stability of immobilized lipase
2
Activated APP membranes (1.0 g, 1.0 cm ) were equilibrated in phosphate buffer
(
(
50 mM, pH 7.0) for 2 h, and transferred to the same fresh medium containing lipase
The operational stability of the immobilized lipase was examined under batch
operation mode at 35 C at intervals of 30 min. After each activity measurement,
the immobilized lipase was separated from medium and washed three times with
◦
50 mg, 2 mg/ml, <3500 units). Immobilization of Candida rugosa lipase on the APP
◦
membrane was carried out at 4 C for 20 h, while continuously stirring the reaction
Fig. 1. Schematic representation of chemical route for the enzyme immobilization.

3
74
G. Bayramo g˘ lu et al. / Process Biochemistry 46 (2011) 372–378
phosphate buffer (5.0 ml, 0.1 M, pH 7.0) and then fresh reaction medium was intro-
duced onto the immobilized lipase. By this way, the next activity measurement was
carried out.
2.8. Storage stability
The storage stability of free and immobilized lipase were determined after stor-
◦
age in phosphate buffer (0.1 M, pH 7.0) at 4 C during 8 weeks. The residual activities
were then determined as described above and activity of the each enzyme was
expressed as percentages of its residual activity compared to the initial activity.
2.9. Enzymatic synthesis of green apple flavor and repeated use of immobilized
lipase
An industrially important ester (i.e., ethyl valerate; green apple flavor) was
synthesized in screw-capped flasks (20 mL) using immobilized lipase on the APP
2
membranes (10 cm ) for both solvent-free medium and hexane medium. For solvent
free system, the enzymatic esterification reactions were carried out with various
valeric acid/ethyl alcohol molar ratios (i.e., 0.5:1.0, 1.0:1.0, 2.0:1.0 and 3.0:1.0 con-
◦
taining of 20 ␮L distilled water) on an orbital shaker at 150 rpm and at 40 C for
2
4 h [25]. For hexane medium, ester synthesis reaction was carried out in n-hexane
Fig. 2. FTIR spectra of (a) polypropylene chloride and (b) aminated polypropylene.
(
15 mL) containing 0.1 mol valeric acid and 0.1 mol ethyl alcohol with shaking
◦
at 150 rpm at 40 C. Along the 24 h reaction, the amount of remaining acid was
determined by titrimetric method using 50 mM NaOH and phenolphthalein as an
indicator. The conversion (%) of ester synthesis was calculated based on the con-
version of the valeric acid to ester after a given time. The initial reaction rate was
determined in the linear region. All of the experiments were carried out in tripli-
cate. The control experiments (no enzyme) were performed and it was observed
that ester yield is less than 2%. After any run, the enzyme-membranes were washed
with phosphate buffer (0.1 M, pH 6.5) and reintroduced into a fresh medium, and
was repeated up to 5 cycles for ester synthesis.
adsorption band at 1628 cm 1 representing N–H bending, is due to
hexamethylene diamine bonded to the polymer backbone.
Scanning electron microscopy (SEM) micrographs of the ami-
nated polypropylene membrane are presented in Fig. 3. The
membrane has a rough surface and non-porous surface structure.
The non-porous surface properties of the membrane would reduce
diffusion limitation of the substrate and product for the enzymatic
hydrolysis and esterification reactions.
The contact polypropylene membrane for immobilization angle
values for water on PPC, APP and APP-lipase immobilized mem-
branes are measured by using sessile drop method. The variation
of the wetting force is extremely sensitive to the surface charac-
−
2
.10. Characterization of polypropylene based membrane
The degree of HMDA incorporation in the synthesized aminated membrane was
determined by measuring the in C, H and N contents with a Laco (Model CHNSO-932)
elemental analyzer. The available amino group’s content of the APP was determined
by potentiometric titration. The APP membrane about 0.1 g was transferred in HCl
teristics since it reflects the effect of functional groups on a surface
layer thickness (less 10 A˚ ) and indirect contact with the liquid phase
◦
solution (0.1 M, 10 ml) and it was then incubated in a shaking water-bath at 35
C
for 6 h. After this reaction period, the final HCl concentration in the solution was
determined by a potentiometric titration with 50 mM NaOH solution. The FTIR spec-
tra of the membrane samples were obtained by using an FTIR spectrophotometer
[
26,27]. The PPC, APP and APP-lipase membranes gave quite differ-
ent surface characteristics depending on the presence of functional
groups. The polypropylene membrane has a hydrophobic surface
(
Shimadzu, FTIR 8000 Series, Japan).
The surface morphology of the APP membrane was observed by scanning elec-
◦ ◦
<90 ) with a water contact angle value of 106 . The change of the
(
tron microscopy (SEM). The dried APP membrane was coated with gold under
reduced pressure and their scanning electron micrographs were obtained using a
JEOL (Model JSM 5600; Japan).
contact angles after amination of the PPC membrane shows that the
◦
hydrophilicity of the surface is increased (78 ). The improvement
Contact angles to water of the PPC, APP and APP-lipase membranes were mea-
sured using sessile drop method at ambient temperature by using a digital optical
contact angle meter Phoneix 150 (Surface Electro Optics, Korea).
of the hydrophilicity of the APP membrane surface caused by the
incorporation of amino groups on the membrane surfaces was used
as a functional component for enzyme coupling after glutaralde-
hyde activation. The change of contact angles after immobilization
◦
3
. Results and discussion
of lipase shows that the hydrophilicity of the surface (67 ) is fur-
ther increased with respect to APP membrane surface. It should be
noted that the hydrophilic nature of the APP surface induces less
conformational changes on the enzyme three-dimensional struc-
3
.1. Characterization studies
In this work, reactive polypropylene based membrane was pre-
pared sequentially in two steps: (i) in the first step, polypropylene
chloride membrane was aminated by using hexamethylenedi-
amine; (ii) then, the aminated membrane was prepared by the
conventional casting method. The total amino group content of
the aminated polypropylene membrane was determined by ele-
mental analysis, and was found to be 6.72 mmol/g. The level of
available surface amino groups of membrane was determined by
using potentiometric titration, and was found to be 5.28 mmol/g
membrane.
The chemical structure of polypropylene chloride (PPC) and
aminated polypropylene (APP) samples were analyzed using a FTIR
(
Fig. 2). Among the characteristic vibrations of PPC is the adsorp-
−1
tion bands at ∼724 cm arising from stretching vibration of C–Cl
−1
bonds. The stretching vibration at 2925 cm represents C–H bands
−
1
in both PPC and APP. The peaks at 1454 and 1371 cm are assigned
as bending of C–H and stretching of C–C bonds. The FTIR spectra
of APP have characteristic broad stretching vibration of –NH and
−1
–
NH2 bands at between 3404 and 3300 cm . The most important
Fig. 3. SEM micrograph of the aminated polypropylene membrane.

G. Bayramo g˘ lu et al. / Process Biochemistry 46 (2011) 372–378
375
1
00
100
8
6
4
2
0
0
0
0
0
8
6
4
2
0
0
0
0
0
Free Lipase
Immobilized Lipase
2
4
6
8
10
0
4
8
12
16
20
24
pH
Time (h)
Fig. 5. Effect of pH on the free and immobilized lipase activity; the relative activities
at the optimum pH were taken as 100% for free and immobilized lipase, respectively.
Fig. 4. Effect of coupling time on the immobilization efficiency of lipase on the
The free and immobilized lipase activity was assayed at different pH values between
aminated polypropylene membrane. The reaction conditions: lipase concentration;
◦
4
.0 and 9.0 and at 35 C for 30 min.
◦
2
mg/ml, temperature; 4 C; medium pH; 7.0 (0.1 M, phosphate buffer), time; 20 h.
3.3. Effect of pH and temperature on the free and immobilized
lipase activity
ture than that of the PPC hydrophobic surface. For this reason,
an orientation of amino acid residues, which influences the wet-
ting by water is more likely in the case of a hydrophilic surface
The effect of pH on the activity of free and immobilized lipase
in olive oil hydrolysis was determined in the pH range 4.0–9.0. The
optimum pH of the free enzyme was 7.0. The optimum pH value
of the immobilized lipase was shifted 0.5 unit to the basic region
(pH 7.5). This shift depended on the method of immobilization
as well as the structure of the matrix (Fig. 5). The pH profile of
the immobilized enzyme was much broader with respect to the
free enzyme, probably due to the product fatty acid, forming lay-
ers around the immobilized enzyme. It should be noted that the
shift to neutral and basic region of the optimal pH upon immo-
bilized could be expected as a result of the diffusional constraint
of the support retaining a higher concentration of enzyme prod-
uct, fatty acids, on the surface of the membrane that immobilized
lipase present. Thus, the microenvironment around the immobi-
lized lipase was more acidic than that of the bulk solution. These
results could probably be attributed to the stabilization of immo-
bilized lipase molecules resulting from multipoint attachment on
the surface of the APP membrane by covalent bonds, which limited
the transition of enzyme conformation against the change of pH.
Other researchers have reported similar observations upon immo-
bilization of lipase and other enzymes [32–34].
[
28,29].
3
.2. Effect of coupling time on the immobilization efficiency
Glutaraldehyde can react with several functional groups of pro-
teins, such as amine, thiol, phenol, and imidazole because the most
reactive amino acid side-chains are nucleophiles [1,30]. Most pro-
teins contain many lysine residues, usually located on the protein
surface (i.e., exposed to the aqueous medium) because of the polar-
ity of the amine group. In addition, lysine residues are generally not
involved in the catalytic site, which allows moderate covalent link-
age to preserve protein conformation and thus biological activity
[
31].
As observed in Fig. 4, an increase in the coupling time
with activated support and lipase led to increase in extent of
immobilization, but this relation leveled off after 12 h. Thus,
a maximum enzyme loading of 76 ␮g/cm2 was observed. No
enzyme leakage was observed during washing of the freshly
prepared enzyme immobilized membrane or during batch oper-
ation mode. This indicates that the immobilization process was
irreversible.
The attachment of 6-carbon and 5-carbon atom spacer-arms on
the PP based membrane surface (during amination with hexam-
ethylene diamine and activation with glutaraldehyde, respectively)
could prevent undesirable side interactions between the large
enzyme molecule and the support. In this way, all areas of the
immobilized lipase could become fully accessible to its large sub-
strate olive oil. The conditions for lipase activity assay were the
same for the free and the immobilized preparations as described
above. The immobilization of lipase through amino groups via glu-
taraldehyde coupling onto on the PP based resulted in a protein
The effect of temperature on the free and immobilized enzyme
activity was investigated in phosphate buffer (0.1 M, pH 7.0) in the
◦
temperature range 20–60 C. The apparent temperature optimum
◦
for free enzyme was about 35 C, while that for the immobilized
◦
enzyme and the form stabilized by immobilization was about 40 C
(Fig. 6). In the literature, most immobilized lipases exhibited higher
optimum temperature values than their free counterpart [35,36].
The multipoint covalent attachment of the lipase molecule on the
membrane surface via glutaraldehyde coupling could reduce the
conformational flexibility of the enzyme and might result in higher
activation energy for the molecule to reorganization the proper
conformation for the binding to its large substrate (i.e., oil olive).
Thus, the immobilized lipase showed its catalytic activity at a higher
reaction temperature compared to that of the free counterpart.
2
loading (76 ␮g protein/cm membrane) and with an activity yield
(
2
69.69 U/cm membrane). The relative activity is the effective activ-
ity after immobilization referred to the activity that the bound
protein amount would have in solution. A low relative activity
indicates that much of the protein was apparently immobilized
in a non-active form. In this work, the recovered activity of the
immobilized lipase was around 76%.
3
.4. Kinetic parameters
The Km value is known as the criterion for the affinity of enzymes
to substrates, and lower value of Km represents the higher affinity

3
76
G. Bayramo g˘ lu et al. / Process Biochemistry 46 (2011) 372–378
100
100
8
6
4
2
0
0
0
0
0
8
6
4
2
0
0
0
0
0
0
2
4
6
8
10
12
Number of cycles
Fig. 7. Operational stability of the immobilized lipase during olive oil hydrolysis
in repeated use. The free and immobilized lipase activity was assayed at 35 C at
intervals of 30 min and at pH 7.0.
◦
Free Lipase
Immobilized Lipase
the catalytic efficiency of an enzyme–substrate pair (Vmax is the
maximum enzymatic reaction rate). In this study, the catalytic effi-
ciencies (Vmax/Km) of the free and immobilized lipase were found
to be 319.3 and 88.2, respectively. The catalytic efficiency of lipase
was decreased about 3.6-fold upon immobilization.
Lipases have a higher level of hydrophobicity than conven-
tional proteins, and the total percentage of hydrophobic amino acid
residues (i.e., Ile, Leu, Val, Met, Tyr, Phe) in lipases isolated from
various microbial sources is varied between 28% and 33% [42]. The
lipases have inherent affinity toward hydrophobic media, and the
hydrophilic/hydrophobic nature of the aminated polypropylene
membrane supports could provide a proper micro-environment
for lipase, and thus, reasonable retained immobilized lipase activ-
ities were obtained with the polypropylene based membrane. The
retained lipase activity was obtained up to 76% in this study, and is
comparable with the related literature [43–45].
10
20
30
40
50
60
70
Temperature (°C)
Fig. 6. Effect of temperature on the free and immobilized lipase activity; the relative
activities at the optimum temperature were taken as 100% for free and immobilized
lipase, respectively. The free and immobilized lipase activity was assayed at different
◦
temperatures between 20 and 60 C and at pH 7.0 for 30 min.
between enzymes and substrates. In this work, kinetic constants
of free and immobilized lipase, i.e., apparent Km and Vmax values
were determined by using tributyrin as substrate. The activities of
the free and immobilized lipase for various concentrations of the
substrate (5–50 mM) were plotted in Lineweaver–Burk plots, and
apparent Km and Vmax values were calculatedfrom the intercepts on
x- and y-axis, respectively. The kinetic data for hydrolysis of tribu-
tyrin was fitted to the Michaelis–Menten equation. For the free and
immobilized lipase the apparent Km values were found to be 2.9 and
3.5. Operational stability in lipid hydrolysis
8
.4 mM, respectively. The apparent Km value of immobilized lipase
was the same order of magnitude, and was only 2.89 times higher
than that of the free enzyme. Thus, the hydrophilic modification of
hydrophobic polypropylene based membrane with hexamethylene
diamine and glutaraldehyde seemed to be the major factor for the
better affinity leading to the lower value of apparent Km for immo-
bilized lipase. The Vmax values for the free and immobilized lipase
were calculated as 926 and 741 U/mg enzyme, respectively. The
Vmax value of the immobilized enzyme decreased about 1.25-fold
compared to the free enzyme.
Several reasons can account for the variations of the Vmax values
of the enzyme upon immobilization [37–41]. These variations are
attributed to several factors such as the covalent attachment of the
enzyme molecule on the membrane surface might have induced
an inactive conformation to the enzyme molecules. It should be
noted that the immobilization process does not also control the
proper orientation of the immobilized enzyme on the support. This
improper fixation and/or the change in the property of the active
sites might hinder the active site for binding of substrates (i.e.,
tributyrin) to the immobilized lipase molecules.
The reusability of an immobilized enzyme is of key impor-
tance for industrial applications, and an increased stability could
make the immobilized enzyme more advantageous than its free
counterparts. Operational stability of the immobilized lipase was
◦
determined for successive batch reactions at 35 C. The results
presented in Fig. 7 shows that the same activity values to lipid
hydrolysis were obtained within the first 5 cycles. After this, a
steady decrease in the oil hydrolysis rate was observed, and this
loss reached 19% after 12 cycles of batch operation, possibly result-
ing from the inactivation of lipase upon use. This result showed
that lipase immobilized on the polypropylene based membrane
could be used successfully for industrial applications requiring
long-term reaction stability. Thus, this immobilization method led
to increased enzyme reusability.
3.6. Thermal stability
The effect of temperature on the stability of the free and immobi-
lized lipase is shown in Fig. 8. The pattern of heat stability indicated
that a smaller rate of thermal inactivation was observed for the
The efficiency factor ꢀ can be calculated from the maximum
reaction rates of the immobilized enzyme over that of the free
counterpart:
immobilized lipase on membrane than that of the free enzyme. At
◦
5
5 C, the free enzyme lost all its initial activity after a 120 min of
heat treatment, while the immobilized enzyme showed significant
resistance to thermal inactivation (retaining about 73% of its initial
activity after the same period). At 65 C, the free lipase lost all its ini-
vimmobilized
ꢀ
=
vfree
◦
where vimmobilized and vfree was the reaction rate of the immobi-
lized and free enzyme, respectively. From this calculation, PP based
membrane enzyme system provided an efficiency factor of 0.8 for
the immobilized lipase. The ratio Vmax/Km defines a measure of
tial activity after 45 min heat treatment. Under the same conditions,
the immobilized lipase retained about 55% of its initial activity. As
in this figure, the free enzyme at 55 and 65 C led to a steep loss of
◦
catalytic activity compared to immobilized counterpart, which is

G. Bayramo g˘ lu et al. / Process Biochemistry 46 (2011) 372–378
377
1
00
40
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
35
30
2
2
1
1
5
0
5
0
5
0
Free Lipase (55 °C)
Free Lipase (65 °C)
Immobilized-Lipase (55 °C)
Immobilized-Lipase (65 °C)
0
1
2
3
4
Valeric acid / ethyl alchol molar ratio
0
50
100
150
Fig. 9. Effect of the molar ratio of acid:alcohol in the non-solvent system on the
ester synthesis efficiency. The enzymatic esterification reaction conditions: valeric
acid: ethyl alcohol molar ratios (i.e., 0.5:1.0, 1.0:1.0, 2.0:1.0 and 3.0:1.0 containing
Time (min)
◦
Fig. 8. Thermal stability of the free and immobilized lipase at two different tem-
peratures. The results of thermal stability are presented in a normalized form with
the highest value of each set being assigned the value of 100% activity. The free and
of 20 ␮L distilled water) at 40 C for 24 h.
◦
alcohol molar ratio, a large increase in alcohol concentration may
promote the binding of alcohol molecules to the lipase, during the
first reaction step, competing with the acid. As a result, a decrease
in the amount of bound acid occurs. Thus, this situation would lead
to a decrease in the reaction rates, since the reaction will be limited
by the amount of acid in the vicinity of the enzyme [13].
immobilized lipase activity was assayed at 35 C at pH 7.0.
typical of most enzymes [1,46], whereas a marked enhancement of
thermal stability was achieved with enzyme immobilization since
no activity decay was observed after one and half hour’s exposure
◦
at 55 C. These results suggest that the thermostability of immobi-
The synthesis of ethyl valerate by immobilized Candida rugosa
lipase was also studied in hexane medium. The ethyl valerate syn-
thesis experiments were carried out under 1:1 acid/alcohol molar
ratio. The amount of synthesized ethyl valerate was 67.2% in the
presence of n-hexane. The amount of the synthesized ethyl valerate
was increased in the presence of n-hexane compared to a solvent-
free system. As reported previously, the presence of an organic
solvent can shift the equilibrium towards ester synthesis by a total
transfer of ester into the organic phase [48]. Similar results related
to the increment in the amount of ester synthesized in organic
medium were reported in literature earlier. For example, Karra-
Chaabouni et al. [49] produced ethyl valerate and hexyl acetate in
hexane, heptane and solvent-free medium. They found remarkably
higher ester yield in both hexane and heptane medium than those
obtained in the absence of organic solvents.
lized lipase becomes significantly higher at higher temperature. If
the heat stability of enzymes increased upon immobilization, the
potential application of these enzymes would be extended.
3.7. Storage stability
Enzymes are not stable during storage in solutions and their
activities decrease gradually by the time. Free and immobilized
lipase preparations were stored in phosphate buffer (1.0 M, pH 7.0)
◦
at 4 C and activity measurements carried out for a period of 8
weeks (data not shown). The free enzyme lost its all-initial activity
within 4 weeks. Immobilized preparations lost 36% activity dur-
ing the same period. The immobilized lipase preserved its initial
activity more than 67% at the end of 8 weeks storage period. The
enhancement in stability provided by the present immobilization
method is more than two-fold. Thus, the covalent immobilization
definitely appears to hold the enzyme in a stable position in com-
parison to the free counterpart.
Operational stability of the immobilized lipase was determined
◦
for 5 successive batch operations at 25 C in solvent free system and
in hexane. In solvent free system, the immobilized lipase remained
almost the same as the original activity after 5 cycles. In hex-
ane medium, a steady decrease in ester synthesis capability of the
immobilized lipase was observed, and this loss reached to about
3.8. Synthesis of green apple flavor by immobilized lipase
1
8% after 5 cycles of batch operation (data not shown). This activity
The effect of acid to alcohol molar ratio (i.e., valeric acid:
loss could be a result of denaturation effect of hexane.
ethyl alcohol molar ratio (i.e., 0.5:1.0, 1.0:1.0, 2.0:1.0 and 3.0:1.0)
was investigated by using immobilized Candida rugosa lipase in
a solvent-free system. The amount of synthesized ethyl valerate
increased from 17.6% to 34.8% with increasing acid to alcohol molar
ratio from 1/2 to 2, respectively. A maximum conversion of 34.8%
was obtained with a molar ratio of 2. It has been reported that high
alcohol concentrations may slow down the reaction rates [13,25].
On the other hand, as shown in Fig. 9, when the molar ratio of acid:
alcohol was further increased, the conversion (%) decreased, which
could be due to the inhibition of the lipase activity by high valeric
acid concentration. As previously reported, in the lipase-catalyzed
esterification reaction, the first step consists of the preferential
binding of the acid molecule to the enzyme molecule [47]. At high
4. Conclusion
One of the most important aims of enzyme technology is to
enhance the conformational stability of the enzyme. The extent
of stabilization depends on the enzyme structure, the immobiliza-
tion methods, and type of support. In the present study, a new
polypropylene based hydrophobic support with functional amino
groups was prepared by modification of PPC with hexamethylene
diamine. The aminated membrane was used for immobilization of
lipase via glutaraldehyde coupling. The properties of the membrane
seem to provide an adequate approach for the immobilization of

3
78
G. Bayramo g˘ lu et al. / Process Biochemistry 46 (2011) 372–378
lipase based on its hydrophobic properties. The immobilized lipase
also exhibited a satisfactory performance for olive-oil hydrolysis
in the activity tests performed in a batch mode. The immobilized
lipase was more stable during operation and storage compared
with free lipase. Finally, the ethyl valerate synthesis was conducted
in a solvent-free system and in n-hexane medium by using the
immobilized lipase. In the case of solvent free system, high conver-
sion of 34.8% was achieved with an addition of 20 ␮L of water, using
[22] Raghavendra T, Sayania D, Madamwar D. Synthesis of the ‘green apple ester’
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2
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