Production of aroma esters by immobilized Candida rugosa and porcine pancreatic lipase into calcium alginate gel

Article abstract of DOI:10.1016/j.molcatb.2009.04.013

Candida rugosa lipase (CRL) and porcine pancreatic lipase (PPL) were immobilized into calcium alginate (Ca-Alg) gel beads by means of entrapment and were used to produce three industrially important flavour esters, namely isoamyl acetate (banana flavour), ethyl valerate (green apple flavour) and butyl acetate (pineapple flavour). Immobilization conditions were optimized in terms of sodium alginate (Na-Alg) and CaCl₂ concentrations by determination of the entrapped enzyme amount as well as by esterification of 4-nitrophenol and acetic acid. The best results were obtained at 2.5% Na-Alg and 2.5 M CaCl₂ for CRL while at 2.5% Na-Alg and 2.0 M CaCl₂ for PPL. On carrying out flavour syntheses in solvent-free medium and also in hexane medium, higher ester yields were obtained in hexane medium for all esters and both types of lipases. Ester esterification efficiency increased in parallel with both enzyme concentrations at immobilization medium and the immobilized lipase amount in esterification medium. Maximum ester production was observed between 40 and 50 °C for CRL and PPL. Besides, the effect of substrate concentrations on ester conversion was remarkable. The best ester yield was obtained for isoamyl acetate when immobilized PPL was used.

Full text of DOI:10.1016/j.molcatb.2009.04.013

Journal of Molecular Catalysis B: Enzymatic 64 (2010) 140–145
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Production of aroma esters by immobilized Candida rugosa and porcine
pancreatic lipase into calcium alginate gelଝ
Gul Ozyilmaz∗, Esra Gezer
University of Mustafa Kemal, Faculty of Arts & Sciences, Department of Chemistry, 31040 Hatay, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 3 May 2009
Candida rugosa lipase (CRL) and porcine pancreatic lipase (PPL) were immobilized into calcium alginate
(Ca-Alg) gel beads by means of entrapment and were used to produce three industrially important flavour
esters, namely isoamyl acetate (banana flavour), ethyl valerate (green apple flavour) and butyl acetate
(pineapple flavour). Immobilization conditions were optimized in terms of sodium alginate (Na-Alg) and
CaCl2 concentrations by determination of the entrapped enzyme amount as well as by esterification of
4-nitrophenol and acetic acid. The best results were obtained at 2.5% Na-Alg and 2.5 M CaCl2 for CRL
while at 2.5% Na-Alg and 2.0 M CaCl2 for PPL. On carrying out flavour syntheses in solvent-free medium
and also in hexane medium, higher ester yields were obtained in hexane medium for all esters and both
types of lipases. Ester esterification efficiency increased in parallel with both enzyme concentrations at
immobilization medium and the immobilized lipase amount in esterification medium. Maximum ester
production was observed between 40 and 50 ◦C for CRL and PPL. Besides, the effect of substrate concen-
trations on ester conversion was remarkable. The best ester yield was obtained for isoamyl acetate when
immobilized PPL was used.
Keywords:
Candida rugosa lipase
Porcine pancreatic lipase
Calcium alginate
Isoamyl acetate
Ethyl valerate
Butyl acetate
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
processing, production of surfactants and enantiomerically pure
pharmaceuticals [3]. There are several studies about ester syn-
The increasing demand for fragrance and flavour esters used
in the food, cosmetics and pharmaceutical industries makes it
necessary to find alternative ways instead of extraction from their
natural materials which are too scarce or expensive for commercial
use. The industrial ester syntheses are based on direct chemical
esterification of fatty acids with alcohol in the presence of inor-
ganic catalysts at elevated temperatures (200–250 ◦C). However,
these chemical reactions are tedious, non-selective and consume a
large amount of energy. Using enzymes and other biocatalysts for
these synthesizing processes allows products with better odor and
color [1].
thesis in non-aqueous medium using lipase by transesterification
[4–8] and esterification reactions [9–18] in solvent-free medium
[11,13,17,19,20]orin organic solvent medium [3,6,21,22]. Main prob-
lem appearing with organic medium when free lipase is used is
denaturation and also aggregation, which prevents homogeneity in
reaction medium. This problem can be solved by using immobilized
form of lipase [23]. Immobilization of enzymes can offer several
advantages including its reuse, ease in application of both batch
and continuous systems, possibility of better control reactions,
ease in removal from the reaction medium and improved stability
[24].
Lipases (triacylglycerol hydrolase, EC 3.1.1.3) are a family of
enzymes that are in their natural environment catalyze the hydrol-
ysis of fats. However, under appropriate working conditions, lipases
have shown to be very active catalysts in esterification, trans-
esterification and alcoholysis reactions [2]. Lipases have been
widely used for biotechnological applications in dairy industry, oil
The aim of this work is to produce three commercially impor-
tant flavour esters, namely isoamyl acetate (banana flavour), ethyl
valerate (green apple flavour) and butyl acetate (pineapple flavour)
by using Candida rugosa lipase (CRL) and porcine pancreatic lipase
(PPL) immobilized into calcium alginate gel (Ca-Alg). The effect
of sodium alginate and CaCl2 concentration on immobilization
was investigated. In esterification reactions firstly flavour ester
synthesis was carried out at solvent-free system and in hexane
medium to compare the effect of non-polar solvent. Also investi-
gated were the effects of various flavour synthesizing parameters
such as enzyme concentration at immobilization, the amount of
immobilized enzyme, temperature, concentration of the substrates
and the reaction time.
ଝ
This study was presented at Enzyme Engineering Symposium IEES’08 in
Kusadasi, Turkey between October 1 and 5, 2008.
∗
Corresponding author. Fax: +90 326 2455867.
E-mail address: gozyilmaz@gmail.com (G. Ozyilmaz).
1381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.04.013

G. Ozyilmaz, E. Gezer / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 140–145
141
2. Experimental
measured at 410 nm. Concentration of 4-NP was determined by
standard curve of 4-NP at different concentration rates prepared
in hexane and extracted with 3.5 ml of 25 mM NaOH solution.
Concentration difference of 4-NP between non-catalyzed and cat-
alyzed medium was used to calculate the amount of 4-NP which
participated in esterification reaction. Activity was expressed as
␮mol 4-NP min−1 mg protein−1. All reactions were repeated three
times.
2.1. Material
C. rugosa lipase (solid form 1170 U mg−1), porcine pancreatic
lipase (solid form 329 U mg−1), isoamyl alcohol (IAA), butyl alcohol
(BA), ethyl alcohol (EA), glacial acetic acid (AA), valeric acid (VA),
sodium alginate (Na-Alg), CaCl2 anhydrous, 4-nitrophenol (4-NP),
NaOH, HCl, hexane and all other chemicals were purchased from
Sigma Chemical Co., St. Louis, MO, USA.
2.2.2.3. Enzyme leakage. The absorbance value of hexane (5 ml)
containing 0.25 g Ca-Alg beads was measured at 280 nm periodi-
cally during 180 min, where pure hexane was used as blank. The
leakage percentage of enzyme from Ca-Alg beads to hexane was
calculated by the equation given below:
2.2. Method
2.2.1. Lipase immobilization into calcium alginate gel
A 100 ml of Na-Alg solution containing CRL (or PPL) was dripped
with syringe into 200 ml of CaCl2 solution at 4 ◦C. As soon as the
mixed solution was dripped into CaCl2 solution, calcium alginate
(Ca-Alg) gels were formed by cross-linking. After 2 h of hardening
time at 4 ◦C, Ca-Alg beads were separated from CaCl2 solution by
vacuum filtration and washed twice with 25 ml of distilled water.
Filtered CaCl2 solution and washing solutions were then mixed
and the total volume of the filtrate was measured. The same pro-
cedure was repeated in the absence of CRL (or PPL), upon which
lipase-free Ca-Alg gel was obtained. The absorbance values of fil-
trate solutions were measured at 280 nm to determine the amount
of non-immobilized enzyme. To convert the absorbance value to
enzyme concentration value, standard curves of enzymes at 280 nm
were previously constituted using CRL and PPL solutions prepared
at different concentrations between 0.01 and 2.00 mg ml−1. Ca-Alg
gels were dried at room temperature open the atmosphere until a
certain weight and samples were then stored in screwed bottles at
4 ◦C.
CLVH
enzyme leakage (%) =
× 100
mL
where CL is the enzyme concentration of hexane solution, VH the
volume of hexane (5 ml), and mL is the amount of the enzyme pro-
tein in 0.25 g of Ca-Alg beads.
2.2.3. Flavour ester synthesis
Three different industrially important esters were synthesized:
isoamyl acetate (IAAc), ethyl valerate (EV) and butyl acetate (BAc)
using CRL or PPL entrapped in Ca-Alg.
Flavour esters were firstly synthesized in solvent-free medium
and in hexane medium to determine the proper medium. To this
end, 1 ml of acid and alcohol mixture at 1:1 molar ratio was
used to synthesize ester with 0.1 g of immobilized enzyme in
a temperature-controlled orbital shaker. After 2.5 h of reaction
time, the amount of remaining acid was determined by titrimet-
ric method using 0.25 M NaOH. Afterwards, studies were repeated
by adding 1 ml of hexane into substrate mixture before starting the
reaction.
For the investigation of the ester synthesis parameters, reactions
were carried out in screw-capped bottle using 0.1 g of Ca-Alg beads
and 10 ml substrate mixture of 25 mM acid (AA or VA) and 25 mM
alcohol (IAA, BA or EA) in hexane with shaking at 200 rpm. At the
end of the 1 h of reaction time, unconsumed acid was extracted
twice by 10 ml of 25 mM NaOH. Then, hexane medium was washed
twice by water. Extraction and washing solutions were collected in
an Erlenmayer and the concentration of remaining NaOH was deter-
mined by back titration using 25 mM HCl solution and bromocresol
green as indicator. Reactions were repeated three times and the
difference of HCl volumes between enzyme containing Ca-Alg and
enzyme-free Ca-Alg was used to calculate the amount of acid sub-
strate participating in the esterification reactions.
2.2.2. Optimization of immobilization parameters
Immobilization was carried out making use of Na-Alg and CaCl2
solutions at different concentrations. Na-Alg concentration was
changed to 1.0–1.5–2.0–2.5–3.0% while CaCl2 and enzyme concen-
tration were kept constant as 2.0 M and 2 mg ml−1, respectively.
Three parameters were investigated for each sample including the
amount of entrapped enzyme, esterification activity by using 4-
nitrophenol and acetic acid and also leakage of the entrapped
enzyme to hexane medium during 180 min. After finding the best
result for Na-Alg, CaCl2 concentration was optimized by changing
from 1.0 to 2.5 M at optimal Na-Alg concentration.
2.2.2.1. The amount of entrapped enzyme. The amount of entrapped
enzyme was calculated according to equation given below:
CoVo − CmVm
CE
=
mE
where CE is the amount of entrapped enzyme per gram of Ca-Alg
(mg g Ca-Alg−1), Co the lipase concentration of Na-Alg solution used
in immobilization (mg ml−1), Vo the volume of Na-Alg solution (ml),
Cm the enzyme concentration of total filtrate (mg ml−1), Vm the vol-
ume of filtrate (ml) and mE is the amount of Ca-Alg beads (g) after
drying.
2.2.3.1. The effect of the esterification parameters. Ester synthesis
was carried out by Ca-Alg beads prepared with CRL or PPL at
various concentrations ranging from 1 to 5 mg ml−1. After deter-
mining the best concentration of CRL and PPL at immobilization, the
effects of acid and alcohol concentrations on ester production were
investigated by two sets of reactions. Firstly, alcohol concentration
was kept constant as 100 mM and acid concentration was selected
between 10 and 100 mM. Remaining acid was extracted twice by
20 ml of 50 mM NaOH and back titration was carried out using 0.1 M
HCl. Secondly, the same type of experiment was repeated with dif-
ferent alcohol concentrations between 10 and 100 mM at optimum
acid concentration for each lipase and flavour ester.
2.2.2.2. Esterification activity for 4-nitrophenol and acetic acid.
Esterification activity was measured by spectrophotometric
method described in a previous study by Ozyilmaz [25] in detail.
Reaction commenced on addition of 0.2 g Ca-Alg beads to 2 ml of
hexane containing 1 mM 4-NP and 50 mM AA and continued for
15 min in a shaker. On completion of reaction time, 1 ml solution
was removed from the reaction mixture and added to 3.5 ml of
25 mM NaOH solution to recover the remaining part of 4-NP by
vortexing for 30 s. The absorbance value of aqueous phase was
Different effects of the amount of Ca-Alg beads (0.05–0.4 g), of
the temperature (30–70 ◦C), and of reaction time (1–48 h) were
studied to find out the best working conditions for flavour ester
synthesis.

142
G. Ozyilmaz, E. Gezer / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 140–145
3. Results
it was found that 34%, 19% and 33% more ester were produced in
hexane medium than those in solvent-free medium for IAAc, EV
and BAc, respectively. The same tendency was observed for PPL
synthesis: 14%, 42% and 6% more yield were obtained in hexane
medium for IAAc, EV and BAc, respectively. Similar results related
to the increment in ester yield in organic medium were reported in
literature earlier. Chaabouni et al. [11] 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.
These results showed that the presence of a solvent could shift the
equilibrium towards ester synthesis possibly by a total transfer of
ester into the organic phase. Similarly, Been Salah et al. [21], who
synthesized butyl acetate in tert-butanol, chloroform, hexane and
heptane, observed that hydrophobicity of organic solvent greatly
affected and improved the esterification activity. They found higher
conversion rates in heptane and in hexane media. Bezbradica et al.
[19] synthesized pentyl, heptyl and geranyl esters of butyric acid
in solvent-free medium and in isooctane medium. At the end of
the 72 h of reaction time, ester conversions were approximately
20% in solvent-free medium and 90% in isooctane medium. How-
ever, when they used carboxylic acid with long chain as oleic acid
instead of butyric acid, conversion rates in solvent-free and in isooc-
tane medium were both nearly 90%. In this study AA and VA which
are carboxylic acid with short chain were used and ester production
proved to be lower in solvent-free medium as cited in the literature.
Subsequent ester synthesis was carried out in hexane medium due
to higher ester productivity in solvent medium.
3.1. Optimization of the immobilization conditions
Because cross-linking between alginate and Ca2+ ions leads to
gelation, the concentrations of Na-Alg and CaCl2 are major param-
eters for enzyme gel entrapment [26]. Therefore, the effects of
alginate and CaCl2 concentration on the entrapment, activity and
enzyme leakage were first investigated and results were summa-
rized in Table 1. While Na-Alg concentration increased, entrapped
lipase amount decreased from 21.9 to 10.3 mg g Ca-Alg−1 for CRL
and from 17.2 to 5.9 mg g Ca-Alg−1 for PPL. Activities of lipase con-
taining Ca-Alg beads measured by spectrophotometric method [25]
and maximum activities were observed at 2.5% Na-Alg concen-
tration for both CRL (0.026 U mg−1) and PPL (0.033 U mg−1). In
case of CaCl2 concentrations, while the Na-Alg concentration was
kept constant at 2.5%, a slight decrease was observed when CaCl2
concentration increased. As in Table 1, there was no significant dif-
ference in the amount of entrapped enzyme. The highest activities
were observed at 2 M CaCl2 as 0.043 U mg−1 and 2.5 M CaCl2 as
0.057 U mg−1 for PPL and CRL, respectively. Enzyme leakage less-
ened in parallel with an increase in Na-Alg and CaCl2 concentration.
At optimum conditions, after 180 min of duration time only 2.24%
and 1.62% of entrapped enzyme leached out of the Ca-Alg to hexane
for CRL and PPL, respectively. These results may have been observed
due to the low solubility of enzymes in organic solvents causing
reduction in the amount of enzyme loss from the gel material [23].
3.2. Flavour ester synthesis
3.2.1. The effect of enzyme concentration at immobilization
Theeffectoftheconcentrationofenzymeusedduringtheimmo-
bilization step was studied and reported in Table 2.
As seen in Table 2, not only the amounts of the entrapped
enzymes but also the esterification activity of Ca-Alg gels increased
To determine the effect of the reaction medium on the esteri-
fication yield, flavour esters were first synthesized at solvent-free
medium and then in hexane medium. When the amounts of acid
substrates used in esterification reaction were compared for CRL,
Table 1
The optimization of immobilization parameters depending on Na-Alg and CaCl2 concentrations.
CRL
PPL
Bound lipasea
Activity
Specific activity
Enzyme
Bound lipase
Activity
Specific activity
Enzyme
leakage (%)
(mg g gel−1
)
(U g gel−1
)
(U mg−1
)
leakageb (%)
(mg g gel−1
)
(U g gel−1
)
(U mg−1
)
CNa-Alg (%)c
1.5
2.0
2.5
3.0
21.9
17.5
13.8
10.3
0.197
0.210
0.359
0.196
0.009
0.012
0.026
0.019
22.48
16.50
6.64
17.2
10.4
7.01
5.9
0.189
0.239
0.231
0.083
0.011
0.023
0.033
0.014
18.71
12.58
7.90
4.58
4.56
CCaCl (M)d
2
1.0
1.5
2.0
2.5
13.7
0.082
0.335
0.467
0.650
0.006
0.025
0.038
0.057
18.45
11.61
6.23
7.8
7.1
6.5
6.2
0.054
0.185
0.279
0.198
0.007
0.026
0.043
0.032
9.26
4.32
1.62
1.15
13.4
12.3
11.4
2.24
a
The amount of entrapped lipase into Ca-Alg gel.
b
c
The leakage of lipase from Ca-Alg gel (0.25 g) into hexane (5 ml) at the end of the 180 min duration time.
Na-Alg concentration was changed while the concentration of CaCl2 was kept constant at 2 M.
CaCl2 concentration was changed while the Na-Alg concentration of was kept constant at 2.5%.
d
Table 2
The effect of enzyme concentration used in immobilization on esterification yield.
Co (mg ml−1
)
CE (mg g Ca-Alg−1
)
Esterification yield of CRL
Esterification yield of PPL
(␮mol ester h−1 g Ca-Alg−1
)
(␮mol ester h−1 g Ca-Alg−1
)
CRL
PPL
IAAc
EV
BAc
IAAc
EV
83
108
168
175
210
BAc
1
2
3
4
5
6.3
12.3
20.9
29.9
31.6
4.8
6.2
8.9
12.9
13.2
100
175
250
275
308
100
165
190
196
200
200
290
375
450
475
150
250
300
325
360
175
250
325
375
400

G. Ozyilmaz, E. Gezer / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 140–145
143
Fig. 1. The effect of the amount of Ca-Alg on the ester yield for (a) CRL and (b) PPL. ꢀ: IAAc, ꢁ: EV, and ꢀ: BAc.
by enzyme amount used in immobilization processes. However,
this increment was not proportional to the amount of enzyme,
because in the presence of high lipase amount in Ca-Alg gel, the
active site may not be in close contact with the substrates and/or
many molecules of the enzyme can aggregate together [11]. Gen-
erally, the increment of ester production lessened after 3 mg ml−1
enzyme concentration. So, immobilization of CRL and PPL was car-
ried out using enzyme concentration at 3 mg ml−1 at subsequent
studies.
while the highest activities were observed at 50 and 45 ◦C for CRL
and PPL, respectively. In the case of BA production, maximum activ-
ities were observed at 45 and 50 ◦C for CRL and PPL, respectively.
As seen, each ester system has different optimal temperatures
even when the same enzyme was used. Similarly, Chaabouni et al.
[11] found optimal temperature for Staphylococcus simulans lipase
immobilized onto CaCO3 as 45 and 37 ◦C for ethyl valerate and hexyl
acetate, respectively. It is safe to say that substrate thermodynamic
properties determine their chemical reactivity and reaction equi-
libria. These properties notably depend on substrate chain-length,
temperature and solvent especially for lipase [27]. Temperature also
has a huge influence on the physical state of substrates such as
solubility, ionization, etc.
3.2.2. The effect of Ca-Alg amount
The effect of Ca-Alg amount ranging from 0.05 to 0.4 g on the
ester yield was studied and results were given in Fig. 1. The pro-
duction of IAAc and EV increased in parallel with Ca-Alg amount
up to 0.2 and 0.3 g, respectively, and then a slow decrease was
observed at higher enzyme amount. In case of BA synthesis, ester
yield was proportional to the amount of Ca-Alg used in hexane
medium. Chaabouni et al. [11] who produced ethyl valerate and
hexyl acetate using different amounts of immobilized lipase onto
CaCO3, reported that yields per milligram of lipase decreased by an
increase in the immobilized amount of lipase.
3.2.4. The effect of substrate concentration
Ester productivity of immobilized CRL and PPL was firstly inves-
tigateddependingonacidconcentrationrangingfrom10to100 mM
at 100 mM constant alcohol concentration and at optimal temper-
atures for each individual ester. As seen in Fig. 3(a) and (b), the
highest amount of BAc was achieved when 25 mM acid was used
for both CRL and PPL. The BAc production decreased sharply in
parallel with acid concentration exceeding 25 mM. IAAc produc-
tion increased with AA concentration up to 75 and 50 mM for CRL
and PPL, respectively. It was observed that IAAc production was
rather low at 100 mM acid. As for EV yield, it increased with VA
concentration up to 100 mM. It was clearly concluded that, at AA
concentration higher than 50 mM, a significant decrease in conver-
sion yield was observed probably due to lipase denaturation by AA
[11,13,21]. After investigating the effect of the acid concentration,
alcohol concentration was changed from 10 to 100 mM at the most
efficient acid concentration for each ester and enzyme. As can be
seen in Fig. 3(c) and (d), the highest yields were obtained for IAAc
at 25 and 75 mM of IAA concentration for CRL and PPL, respectively.
3.2.3. The effect of temperature
The effect of temperature on the progress of the lipase-catalyzed
esterification reactions was studied using 0.1 g of immobilized
enzyme at 8 different temperatures between 30 and 70 ◦C, and
results were given in Fig. 2.
On increasing the temperature from 30 to 40 ◦C, there were
remarkable changes in the conversion profile of the IAAc produc-
tion for both CRL and PPL, and after 40 ◦C the ester yield decreased
dramatically. Moreover, IAAc production was observed at temper-
ature higher than 60 and 70 ◦C for CRL and PPL, respectively. The
minimum effect of temperature on production was observed for EV
Fig. 2. The effect of temperature on the ester yield for (a) CRL and (b) PPL. ꢀ: IAAc, ꢁ: EV, and ꢀ: BAc.

144
G. Ozyilmaz, E. Gezer / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 140–145
Fig. 3. The effect acid concentration on ester yield for (a) CRL and (b) PPL at 100 mM constant alcohol concentration, and the effect of alcohol concentration on ester yield for
(c) CRL and (d) PPL at constant optimal acid concentration. ꢀ: IAAc, ꢁ: EV, and ꢀ: BAc.
Fig. 4. Ester productivity at different reaction times for (a) CRL and (b) PPL. ꢀ: IAAc, ꢁ: EV, and ꢀ: BAc.
While, the highest amount of BAc was obtained at 50 mM of BA
concentration for both CRL and PPL, this value was 75 mM EA for
EV synthesis. The lowest yield was observed for EV synthesis. It can
be seen in Fig. 3(c) and (d) that, PPL was more efficient than CRL to
produce flavour esters at their optimal working conditions.
Although EV productivity increased with VA concentration up
to 100 mM yield increased up to 75 mM, and then decreased in the
case of increment in alcohol concentration. It may be due to more
inhibitor effect of EA than those of IAA and BA [11].
the enzyme in excess of the amount needed for complete hydration
layer may provide some protection from denaturation by organic
solvents, however, this may cause mass transfer problems for sub-
strate. Most probably, increased water may lead to hydrolysis of
ester previously produced [23].
The best result was obtained for IAAc synthesis when PPL was
used. However, the minimum yield was obtained for EV flavour
when CRL was used as catalyst.
4. Conclusion
3.2.5. The effect of reaction time
Ester synthesis was achieved at their optimal conditions at dif-
ferent reaction times between 1 and 48 h and results were given in
Fig. 4. Ester production increased with time up to 10 h of reaction
time and then slightly decreased while using CRL. Similar results
were observed for PPL as production of EV and BAc, except IAAc.
This decrease may be explained by an increment in the amount
of water produced during catalysis. Large amounts of water around
The production of IAAc (banana flavour), EV (green apple
flavour) and BAc (pineapple flavour) was carried out by CRL and PPL
immobilized into Ca-Alg gel. Immobilization conditions were opti-
mized in terms of Na-Alg and CaCl2 concentration in detail. Ester
productions were higher when synthesis was carried out in hexane
medium instead of solvent-free medium. It was clearly shown that,
CRL and PPL have different affinities for different substrates. The

G. Ozyilmaz, E. Gezer / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 140–145
145
best yield was obtained for IAAc by PPL while the yield for EV by
CRL was lowest.
[5] K.B.K. Lee, L.H. Poppenborg, D.C. Stuckey, Enzyme Microb. Technol. 23 (1998)
253–260.
[6] G.D. Yadav, A.H. Trivedi, Enzyme Microb. Technol. 32 (2003) 783–789.
[7] A.P. de los Ríos, F.J. Hernández-Fernández, F. Tomás-Alonso, D. Gómez, G. Víllora,
Flavour Frag. J. 23 (2008) 319–322.
[8] W. Kroutil, L. Hagmann, T.C. Schuez, V. Jungmann, J.P. Pachlatko, J. Mol. Catal. B:
Enzym. 32 (2005) 247–252.
[9] S. Bourg-Garros, N. Razafindramboa, A.A. Pavia, Biotechnol. Bioeng. 59 (1998)
495–500.
Nomenclature
CRL
PPL
IAA
EA
Candida rugosa lipase
porcine pancreatic lipase
isoamyl alcohol
ethyl alcohol
[10] G.D. Yadav, S.B. Dhoot, J. Mol. Catal. B: Enzym. 57 (2009) 34–39.
[11] M.K. Chaabouni, H. Ghamgui, S. Bezzine, A. Rekik, Y. Gargouri, Process Biochem.
41 (2006) 1692–1698.
BA
butyl alcohol
AA
VA
IAAc
EV
BAc
acetic acid
valeric acid
isoamyl acetate
ethyl valerate
butyl acetate
[12] A. Kılınc, M. Teke, S Onal, A. Telefoncu, Prep. Biochem. Biotechnol. 36 (2006)
153–163.
[13] A. Guvenc, N. Kapucu, U. Mehmetoglu, Process Biochem. 38 (2002) 379–
386.
[14] R. Kumar, J. Modak, G. Madras, Biochem. Eng. J. 23 (2005) 199–220.
[15] S.H. Krishna, S. Divakar, S.G. Prapulla, N.G. Karanth, J. Biotechnol. 87 (2001)
193–201.
[16] S.W. Chang, J.F. Shaw, C.K. Yangc, C.J. Shieh, Process Biochem. 42 (2007)
1362–1366.
Na-Alg sodium alginate
Ca-Alg calcium alginate
[17] H. Gonzalez-Navarro, L. Braco, Biotechnol. Bioeng. 59 (1998) 122–127.
[18] J.M. Rodriguez-Nogales, E. Roura, E. Contreras, Process Biochem. 40 (2005)
63–68.
4-NP
4-nitrophenol
Acknowledgement
[19] D. Bezbradica, D. Mijin, S. Siler-Marinkovic, Z. Knevic, J. Mol. Catal. B: Enzym.
45 (2007) 97–101.
[20] H. Abbas, L. Carneau, Enzyme Microb. Technol. 32 (2003) 589–595.
[21] R. Been-Salah, H. Ghamghui, N. Miled, H. Meidoub, Y. Gargouri, J. Biosci. Bioeng.
103 (2007) 368–372.
[22] A. Zaidi, J.L. Gainer, G. Corta, A. Mrani, T. Kadiri, Y. Belarbi, A. Mir, J. Biotechnol.
93 (2002) 209–216.
The authors gratefully acknowledge the financial supporting of
this work by the Scientific and Technical Research Council of Turkey,
TUBITAK (Project No. 107T919).
[23] A.R.M. Yahya, W.A. Anderson, M. Moo-Young, Enzyme Microb. Technol. 23
(1998) 438–450.
References
[24] G. Ozyilmaz, S. Tukel, O. Alptekin, Mol. Catal. B: Enzym. 35 (2005) 154–160.
[25] G. Ozyilmaz, J. Mol. Catal. B: Enzym. 56 (2009) 231–236.
[26] K. Won, S. Kim, K.J. Kim, H.W. Park, S.J. Moon, Process Biochem. 40 (2005)
2149–2154.
[27] L. Vaysse, A. Ly, G. Moulin, E. Dubreucq, Enzyme Microb. Technol. 26 (2000)
421–430.
[1] M.A. Kiss, E. Sefanovits-Banyai, A. Toth, L. Boross, Eng. Life Sci. 4 (2004) 460–464.
[2] M.L. Foresti, M.L. Ferreira, Enzyme Microb. Technol. 40 (2007) 769–777.
[3] A. Hiol, M.D. Jonzo, N. Rugani, D. Druet, L. Sarda, L.C. Comeau, Enzyme Microb.
Technol. 26 (2000) 421–430.
[4] W.D. Chiang, S.W. Chang, C.J. Shieh, Process Biochem. 38 (2003) 1193–1199.