Immobilization of lipase in organic solvent in the presence of fatty acid additives

Article abstract of DOI:10.1016/j.molcatb.2010.08.008

In this study porcine pancreatic lipase (PPL) was covalently immobilized on cross-linked polyvinyl alcohol (PVA) in organic media in the presence of fatty acid additives in order to improve its immobilized activity. The effects of fatty acid additions to the immobilization media were investigated choosing tributyrin hydrolysis in water and ester synthesis by immobilized PPL in n-hexane. Various fatty acids which are also the substrates of lipases in esterification reactions were used as active site protecting agents during the immobilization process in an organic solvent. The obtained results showed that covalent immobilization carried out in the presence of fatty acids as protective ligands improved the hydrolytic and esterification activity of immobilized enzyme. A remarkable increase in activity of the immobilized PPL was obtained when octanoic acid was used as an additive and the hydrolytic activity was increased from 5.2 to 19.2 μmol min^-1 mg^-1 as compared to the non-additive immobilization method. With the increase of hydrolytic activity of immobilized lipase in the presence of octanoic acid, in an analogous manner, the rate of esterification for the synthesis of butyl octanoate was also increased from 7.3 to 26.3 μmol min^-1 g^-1 immobilized protein using controlled thermodynamic water activities with saturated salt solutions. In addition, the immobilized PPL activity was maintained at levels representing 63% of its original activity value after 5 repeated uses. The proposed method could be adopted for a wide variety of other enzymes which have highly soluble substrates in organic solvent such as other lipases and esterases.

Full text of DOI:10.1016/j.molcatb.2010.08.008

Journal of Molecular Catalysis B: Enzymatic 67 (2010) 214–218
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Immobilization of lipase in organic solvent in the presence of fatty acid additives
Taylan K. Ozturk, Ali Kilinc∗
˙
Department of Biochemistry, Faculty of Science, Ege University, 35100 Bornova-Izmir, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 April 2010
Received in revised form 9 August 2010
Accepted 12 August 2010
Available online 18 August 2010
In this study porcine pancreatic lipase (PPL) was covalently immobilized on cross-linked polyvinyl alco-
hol (PVA) in organic media in the presence of fatty acid additives in order to improve its immobilized
activity. The effects of fatty acid additions to the immobilization media were investigated choosing trib-
utyrin hydrolysis in water and ester synthesis by immobilized PPL in n-hexane. Various fatty acids which
are also the substrates of lipases in esterification reactions were used as active site protecting agents
during the immobilization process in an organic solvent. The obtained results showed that covalent
immobilization carried out in the presence of fatty acids as protective ligands improved the hydrolytic
and esterification activity of immobilized enzyme. A remarkable increase in activity of the immobilized
PPL was obtained when octanoic acid was used as an additive and the hydrolytic activity was increased
from 5.2 to 19.2 ␮mol min−1 mg−1 as compared to the non-additive immobilization method. With the
increase of hydrolytic activity of immobilized lipase in the presence of octanoic acid, in an analogous
manner, the rate of esterification for the synthesis of butyl octanoate was also increased from 7.3 to
26.3 ␮mol min−1 g−1 immobilized protein using controlled thermodynamic water activities with satu-
rated salt solutions. In addition, the immobilized PPL activity was maintained at levels representing 63%
of its original activity value after 5 repeated uses. The proposed method could be adopted for a wide
variety of other enzymes which have highly soluble substrates in organic solvent such as other lipases
and esterases.
Keywords:
Porcine pancreatic lipase
Immobilization in organic solvent
Additives
Esterification
Water activity
Fatty acid
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
By using additives such as ligands, substrates, substrate ana-
logues or other components during the enzyme immobilization
The use of organic solvents as reaction media for biocatalytic
reactions instead of conventional aqueous media has proven to be
an extremely useful approach for expanding the range and effi-
ciency of practical applications of lipases and it has been well
documented [1,2]. However, the activity of the reported native
enzymes in anhydrous organic media is lower than in water. To
counter this limitation many efforts have been made to improve
the catalytic activity and stability in non-aqueous media. In many
cases immobilization of lipases has proved to be a useful technique
for enhancing enzyme activities in organic solvents [3].
One of the main drawbacks associated with the use of immobi-
lization methods is the loss of catalytic activity [4]. On the other
hand, immobilization of lipases when used in anhydrous media
warrants the dispersion of the biocatalyst and, in addition, prevents
the aggregation of hydrophilic protein particles [3]. Nevertheless,
the loss of activity of lipases due to the restriction of conformational
changes and formation of rigid structures upon immobilization
remains a great challenge in covalent fixation [5].
process the enzyme’s active center can be protected. In this way,
the active center occupied by a substrate molecule often prevents
a conformational change of the enzyme during covalent binding,
thus preserving its high catalytic activity [6]. In addition, it has
been reported that lipases pretreated with organic solvents yield
an enhancement of both enzyme activity and stability [2,7].
Recently several papers regarding the immobilization of lipases
by adsorption in organic solvents have been published demon-
strating that lipase immobilization carried out in organic solvents
enhanced the enzyme activity and stability as compared to immo-
bilization in aqueous phases [8–11].
In this study porcine pancreatic lipase (PPL) was covalently
immobilized onto cross-linked PVA in an organic solvent in the
presence of a fatty acid substrate. Using an organic solvent pro-
vides effective solubility of fatty acid additives thereby enhancing
the interaction of these compounds with the active center of the
enzyme. Their presence often counteracts conformational changes
of the biocatalyst during covalent immobilization, thus retention
of the activity can be secured. The main idea of this approach using
fatty acids as protective additives is the avoidance of harmful denat-
uration effects during covalent immobilization thereby improving
its efficiency. Various fatty acids can be tested as protective candi-
∗
Corresponding author. Tel.: +90 232 311 1775; fax: +90 232 311 1775.
E-mail address: ali.kilinc@ege.edu.tr (A. Kilinc).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.08.008

T.K. Ozturk, A. Kilinc / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 214–218
215
dates for improved activity recovery. Their effect on the hydrolytic
activity of PPL in water as well as the reverse reaction (esterifica-
tion) in n-hexane with controlled water activity has been studied
in this contribution. In addition, the optimal solvent, alcohol and
fatty acid type as well as the water activity and the temperature
effect on the esterification activity of PPL were also determined.
Esterification activities of enzymes were performed in screw-
capped test tubes with a working volume of 1 ml. Unless otherwise
stated, the typical reaction mixture consisted of 0.1 g immobi-
lized enzyme and 1.0 ml of n-hexane containing octanoic acid
and n-butanol as substrates at 300 mM concentrations. The initial
water activity (aw) of substrates and enzyme samples was pre-
equilibrated to aw = 0.75 with a saturated salt solution of NaCl. The
reaction mixture was incubated at 40 ◦C under constant agitation at
200 rpm in an incubator. Initial reaction rates for esterification have
been calculated using the time-dependent results and expressed
in ␮mol ester released per minute by gram immobilized protein
2. Experimental
2.1. Materials
(␮mol min−1 g−1)
.
Porcine pancreatic lipase (EC 3.1.1.3, Type II, Crude; PPL) was
obtained from Sigma Chem. Co. (St. Louise, USA). Polyvinyl alco-
hol (PVA) and adipoyldichloride were purchased from E. Merck
(Darmstad, Germany). Tributyrin as lipase substrate was obtained
from Sigma Chem. Co. Alcohols (methanol, ethanol, n-propanol,
n-butanol, n-octanol, 2-octanol, iso-propanol) and organic acids
(Butyric (C4), Octanoic (C8), Capric (C10), Lauric (C12), Myristic
(C14) and Palmitic (C16) acids) were purchased from Aldrich and
all were of GC grade. All solvents were likewise of GC grade and all
inorganic salts were of analytical grade.
2.5. The effect of initial water activity
In order to determine the optimal initial water activity for ester-
ification reactions of free and immobilized lipase all solvents and
substrates of the esterification reaction were pre-equilibrated at
25 ◦C for 24 h in closed vessels over appropriate saturated salt solu-
tions at different water activities (aw) including NaOH (aw = 0.0)
LiCl (aw = 0.11), MgCl2 (aw = 0.33), Mg (NO3)2 (aw = 0.53), NaCl
(aw = 0.75) and K2SO4 (aw = 0.97) [14]. The effect of the initial aw on
esterification rates was investigated by using equimolar amounts
of n-butanol and octanoic acid (300 mM) for the synthesis of butyl
octanoate at 40 ◦C in n-hexane.
2.2. Immobilization procedure
Preparation of cross-linked PVA as an immobilization matrix
with low solubility in water and organic solvents has been reported
by Kilinc and Önal [12]. In a typical immobilization reaction one
gram of cross-linked PVA matrix and 200 mg of crude PPL with
45.3 U specific activity (hydrolytic activity) were suspended in
5 ml of anhydrous cyclohexane as immobilization media and then
20 ␮l of adipoyldichloride were added. In order to investigate the
effect of fatty acid additives, these compounds (substrates of the
esterification reaction catalyzed by lipases) were added to the
immobilization media containing the enzyme prior to covalent
binding to the support. Thereafter the substrate-primed enzyme
molecules interact with carriers bearing functional hydroxyl
groups. Following this, multipoint attachment occurs during immo-
bilization by cross-linking the polymer–enzyme conjugate by the
means of adipoyldichloride. For this purpose saturated fatty acids
including C4, C8, C10, C12, C14 and C16 were added to the immobi-
lization media at 0.3 M concentrations 5 min prior to addition of the
covalent linker. The reaction mixture was incubated at room tem-
perature for 30 min with continuous stirring. The solid support was
filtered off by suction and successively washed with acetone, cyclo-
hexane and water, respectively, to remove impurities and unbound
protein. The immobilized enzyme was lyophilized and stored at 4 ◦C
until use.
2.6. GC analysis of fatty acid esters
At the end of the incubation period the reaction mixtures were
cooled and diluted with n-hexane and a 1 ␮L aliquot was injected
in a split mode into a Thermo Finnigan Trace GC Ultra® gas chro-
matograph equipped with a flame-ionization detector. A ZB-WAX
(Zebron®) fused silica capillary column (30 m × 0.32 mm i.d.; film
thickness 0.25 ␮m) was used. Nitrogen was used as carrier gas and
both injector and detector temperatures were set at 250 ◦C. The
initial column temperature of 100 ◦C was held for 2 min and then
raised to 220 ◦C at a rate of 40 ◦C min−1 and then held at 220 ◦C.
The extent of synthesis was calculated based on alcohol injected
and quantified by comparison with standard curves of alcohol. All
analyses were performed in triplicates.
2.7. Determination of molar ratios of substrates for esterification
The effect of substrates concentrations was investigated by
varying the concentration of n-butanol and octanoic acid in the
reaction system from 50 to 500 mM. Esterification reactions were
carried out for a period of 4 h using the optimized assay conditions.
2.3. Protein determination
2.8. The effect of temperature on esterification activity
Protein concentrations were determined by the method of
Lowry et al. [13] by using bovine serum albumin as standard.
The amount of bound protein was calculated from the difference
between the amount of protein introduced into the coupling reac-
tion mixture and the amount of protein present in the filtrate and
washing solutions after immobilization.
The effect of reaction temperature was investigated through
incubation of reaction mixtures (n-hexane as solvent, octanoic acid
and n-butanol as substrates at 300 mM concentrations) at various
temperatures ranging from 30 to 80 ◦C for 4 h.
2.9. Reusability of immobilized PPL
2.4. Hydrolytic and esterification activities of lipase
The immobilized enzyme was used in the standard ester synthe-
sis repeatedly. After each run the reaction media was centrifuged
(1000 rpm, 5 min) and the immobilized PPL was washed with 1 ml
of pre-equilibrated n-hexane (aw = 0.75, NaCl) several times. Then
fresh reaction medium containing 300 mM octaoic acid and n-
butanol in 1 ml n-hexane was added and initial reaction rates were
determined from the product produced after 2 h of reaction.
The hydrolytic activities of free and immobilized lipase were
measured titrimetrically at pH 8.0 at 37 ◦C with a pH-stat (718 Stat
Titrino, Metrohm Ltd., Switzerland), under the standard assay con-
ditions described previously [12], using tributyrin (250 ␮L) as the
substrate in 25 mL, 0.1 M NaCl. The released free fatty acids from
tributyrin were titrated with 0.05 M NaOH and one unit of lipase
activity was defined as 1 ␮mol of fatty acid released per minute.

216
T.K. Ozturk, A. Kilinc / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 214–218
Table 1
Effect of different fatty acid additives (300 mM) on hydrolytic activities and initial reaction rates of esterification reaction of butanol and octanoic acid.
Type of additive
Protein immobilizeda (mg g−1 support)
Hydrolytic activity (␮mol min−1 mg−1
)
Esterification activityb (␮mol min−1 g−1
)
Free PPL
Non-additive
C4
C8
C10
C12
C14
C16
–
39
36
36
37
37
36
37
45.3
5.2
36.8
7.3
11.1
19.2
17.2
14.1
13.9
14.0
13.6
26.2
21.4
17.6
17.5
18.6
a
Total protein offered to 1 g support for immobilization: 64 mg. The yield of immobilized protein for non-additive immobilization is 61%.
b
Esterification activities determined in hexane at 40 ◦C and initial water activities of free and immobilized enzymes pre-equilibrated to aw = 0.75.
3. Results and discussion
tive which is also a substrate. Hence, the active cavity of PPL was
filled with a substrate molecule and as a result the adipoyldichlo-
ride bifunctional covalent linker could not reach and react with the
catalytically important amino acids.
3.1. Immobilization of PPL in organic solvents
In the present work multipoint covalent immobilization of PPL
on cross-linked PVA was carried out in cyclohexane in the presence
of different types of fatty acids which are also substrates of lipases
in esterification reactions. This method is the so-called imprinting
enzyme immobilization in which the active site is protected during
the immobilization process in the presence of a ligand [6]. Most of
the studies reported before, however, involve using soluble deter-
gents or non-soluble additives such as oils before immobilization
or during immobilization carried out in an aqueous environment
[15–18].
Moreover, de Castro et al. immobilized PPL by adsorption
on Celite comparing buffer or n-hexane as an immobilization
medium without any additives. It was also found that the esterifica-
tion activity of organic-solvent immobilized PPL was seven times
higher than in aqueous immobilization (the specific activity was
0.22 ␮mol min−1 g−1 for the synthesis of butyl butanoat in hex-
ane) [10]. de Oliveira et al. showed that CRL immobilization in an
organic solvent not only had higher activity for both hydrolysis and
esterification but also had higher stability [11].
The successful introduction of organic solvents as immobiliza-
tion media prompted our studies using fatty acid substrates as
effective protective additives because of their high solubility in
organic solvents. Consequently, to identify the most effective fatty
acid additive resulting in the highest activity retention, various
fatty acids at different concentrations were assayed. The highest
hydrolytic activities were obtained when the fatty acid concentra-
tion was 300 mM with octanoic acid being the most effective one
(Table 1).
Table 1 shows that all the fatty acid additives improved
the hydrolytic activity of immobilized enzyme in comparison to
the non-additive immobilization method. The enhancement of
hydrolytic activity ranged from 5.2 to 19.2 ␮mol min−1 mg−1 when
octanoic acid was used as an additive. Improvement of the activity
can be explained with the affinity of lipase for the fatty acid addi-
Furthermore, esterification activities of immobilized lipases
were also investigated. In a similar manner enhanced esterifica-
tion activities were observed when fatty acids were added to the
immobilization media. The highest esterification activity was like-
wise obtained when octanoic acid was used as an additive and the
esterification activity increased from 7.3 to 26.3 ␮mol min−1 g−1 for
the synthesis of butyl octanoate (Table 1).
Using fatty acids during immobilization in organic solvents led
to a pronounced increase both of hydrolytic and esterification activ-
ity. There was, however, no significant effect of fatty acid addition
on the amount of protein bound to the carrier. Bound protein yields
were in the case of non-additive immobilization about 61% and in
the presence of fatty acids 57–58% (Table 1).
3.2. Effect of solvent type on esterification activities of lipases
The effect of solvent polarity on the esterification activity of
lipase was investigated. For this purpose initial reaction rates and
esterification yields of immobilized lipases were measured with
various monophasic solvents as reaction media including tetrahy-
drofuran, benzene, n-hexane, iso-octane and acetonitrile.
The initial water activity of reaction conditions was pre-
equilibrated to aw = 0.75. Higher initial specific activities and overall
reaction yields were obtained when n-hexane and iso-octane
were used as solvents with water activities set to 0.75. Initial
rates, log P-values and overall yields after 96 h are represented in
Table 2.
The initial reaction rate and over all yield for butyl octanoate
synthesis was higher in iso-octane and n-hexane when com-
pared to polar solvents. A similar trend correlating enzymatic
activity with log P-values of solvents was also reported by other
lipase-catalyzed esterification reactions [19,20]. Lower enzymatic
activities and reaction yields were obtained in polar solvents due to
their ability to strip off essential water molecules from the enzyme
microenvironment [21–23]. Therefore n-hexane was used for fur-
ther esterification reactions.
Table 2
Effect of solvent polarity on the activity and yield after a 96 h lipase-catalyzed ester-
ification of octanoic acid and n-buthanol; initial water activity was set to aw = 0.75.
3.3. Effect of aw on esterification reactions
Solvent
Log P-valuea
Initial reaction rates
Yieldb
%
(␮mol min−1 g−1
)
The effects of water activity on initial reaction rates were also
investigated. The type of organic solvent and the water content of
the microenvironment on the enzyme surface are essential param-
eters for the synthetic activity of lipases. Critical amounts of water
around the enzyme maintains the enzyme in an active conforma-
tion. In addition, the active site polarity and the thermodynamic
equilibrium of the esterification reaction are directly related to the
water activity [24]. Additional water content of the support and
mass transfer from the reaction mixture are affected by the support
Iso-octane
n-Hexane
Benzene
Tetrahydrofuran
Acetonitrile
4.50
3.50
2.00
0.52
20.19
19.30
9.68
3.41
1.92
95
90
67
13
0.5
−0.35
a
LogP-value was defined as the partition co-efficient of the individual solvent
between water and n-octanol.
b
Yield (%) after a 96 h reaction was calculated as the concentration of the ester
product.

T.K. Ozturk, A. Kilinc / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 214–218
217
Fig. 1. Effect of water activity on initial esterification rates of free and immobilized
PPL in the presence of octanoic acid. Initial rates were determined from the results
of 2 h reactions.
Fig. 2. Effect of substrate concentrations on initial reaction rates of esterification
(40 ◦C; aw = 0.75; n-hexane) (ꢀ) 300 mM n-butanol was kept constant; (ꢁ) 300 mM
octanoic acid kept constant; (ꢂ) molar ratio 1:1.
properties [25]. Low water activities favor in general ester synthesis
over the hydrolysis reaction [26].
3.5. Effect of various alcoholic substrates on esterification
The effect of initial aw on the esterification rate for the synthesis
of butyl octanoate is shown in Fig. 1. With an increase of the initial
aw the rate of enzyme activity for non-immobilized PPL decreased.
Higher values of aw caused aggregation of the enzyme and inhibited
the esterification reaction. In addition, the inhibitory effect of the
aggregation for crude PPL was augmented when the reactions were
carried out as well with lower aw values because of the formation
of water product from the esterification reaction. As a consequence
of aggregation, the reaction yield (data not shown) was lower than
with immobilized PPL despite the high initial reaction rates (Fig. 1).
However, the optimal water activity of immobilized PPL was higher
than aw = 0.75. As is obvious from Fig. 1, the measured initial reac-
tion rates indicate a change of the inhibitory effect of water. High
polarity of the support material is apparently responsible for this
change by binding the water molecules which are essential for
the enzyme’s synthetic activity. Moreover, the esterification yield
of immobilized PPL after 96 h reactions with a starting value of
aw = 0.75 was more than 90% (Fig. 3 and Table 3). This water activ-
ity was used for the determinations of esterification activities of
immobilized PPL throughout this work.
The effect of the primary alcohol chain length was investigated
in the synthesis ofoctanoicacidesters. Low reaction ratesand yields
obtained with methanol, ethanol and n-propanol when compared
to n-butanol and n-octanol (Fig. 3). The inhibition effect of highly
polar short chain alcohols was directly related to their ability to
strip water molecules from the enzyme’s surrounding microen-
vironment. Immobilized PPL did not catalyze the reaction with
secondary alcohols as examined (2-propanol and 2-butanol).
3.6. Influence of fatty acid chain length on esterification
The effect of the chain length of acyl donors on the initial
reaction rates of esterification and their yield with butanol was
investigated. Table 3 displays the molar conversion of butanol as
a function of the fatty acid chain length. The different fatty acids
(C8–C16) resulted in similar molar butanol conversions (93%) after
a reaction period of 96 h. It is well known that crude preparations of
PPL represent a composition of various lipolytic enzymes with dif-
ferent properties in terms of activity and selectivity [27]. Therefore
we could not detect any selectivity improvement for immobilized
PPL in the presence of octanoic acid.
3.4. Effect of substrate concentrations on esterification
3.7. Effect of temperature on the esterification reaction
The substrate concentration has a significant influence on the
initial esterification rates of immobilized lipase [10]. Initial reac-
tion rates of immobilized PPL were determined by using equimolar
amounts of n-butanol and octanoic acid from 50 mM to 500 mM
for a reaction period of 2 h. In Fig. 2 the increase of the initial
reaction rates with increasing substrate concentrations with a sat-
uration maximum at 300 mM are shown. Higher levels of acidic
or alcoholic substrates can change the enzyme microenvironment
removing essential water molecules or acidifying the enzyme’s
aqueous phase.
The temperature has a significant effect on the equilibrium of
the reaction and on the activity and stability of immobilized lipase.
Table 3
Effect of chain length of fatty acids on esterification; aw = 0.75.
Fatty acid
Initial reaction rate
Yield (%)
(␮mol min−1 g−1
)
24-h
48-h
96-h
C8
22.5
15.6
16.4
23.3
31.4
37.7
35.2
34.5
36.6
47.5
66.5
57.2
61.0
66.9
75.2
93.7
93.0
92.1
93.7
94.3
C10
C12
C14
C16
Fig. 3. Effect of different chain length alcohols on initial reaction rates (3.5, 15.6,
16.9, 22.9, and 26.2 ␮mol min−1 mg−1, respectively); 40 ◦C; aw = 0.75.

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T.K. Ozturk, A. Kilinc / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 214–218
fatty acid substrates possessing high solubility in organic solvents
appeared to us as effective protective additives for the prepara-
tion of an immobilized biocatalyst with improved activity. In this
study we present a simple protocol to immobilize PPL in an organic
solvent with retention of high activity.
Our results suggest that using fatty acids during immobilization
in organic solvents lead to a pronounced increase both in hydrolytic
and also esterification activity.
The synthesis of various aliphatic esters was successfully per-
formed with immobilized PPL. There is obviously a great potential
in using fatty acid additives as a means to improve the activity of
the immobilized biocatalyst. The proposed method could also be
applicable for a wide variety of other lipases and esterases acting
on non-soluble substrates.
Fig. 4. Effect of temperature on initial reaction rates of esterification reaction.
Acknowledgements
We thank the Scientific Research Foundation of Ege University,
Izmir, Turkey (BAP-Grant Number Sci-2007-13) for financial sup-
port of this work.
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3.8. Reusability of immobilized PPL
Reusability of an immobilized enzyme without loss of activ-
ity is important for its economic viability. The reusability of the
immobilized enzyme was tested by its repeated use for butyl
octanoate synthesis for several 2 h runs (Fig. 5). There was no sig-
nificant change of its reusability due to the immobilization in the
presence of octanoic acid if compared to the non-additive immo-
bilization mode. Immobilized PPL kept its nearly 63% activity after
five separate runs without any additional water activity control.
The observed loss of enzyme activity caused by the change of the
microenvironment of the enzyme during each run probably may
be due to partial denaturation of the enzyme or losses of critical
amounts of water when the reaction mixture was replaced.
4. Conclusion
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Organic-solvent immobilization of enzymes is a rather rare
application in enzymology. With lipase as an example we studied
for the first time its immobilization in non-conventional solvents
in the presence of fatty acids for substrate imprinting. From the
viewpoint of using organic solvents as an immobilization medium
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