ORIGINAL PAPER
C18 Unsaturated Fatty Acid Selectivity of Lipases During
the Acidolysis Reaction Between Tripalmitin and Oleic, Linoleic,
and Linolenic Acids
Ihsan Karabulut • Gokhan Durmaz •
Ali Adnan Hayaloglu
Received: 11 November 2009 / Revised: 28 May 2010 / Accepted: 28 May 2010 / Published online: 13 June 2010
 AOCS 2010
Abstract The C18 unsaturated fatty acid (UFA) selec-
tivity of three immobilized lipases, namely, Lipozyme TL
IM from Thermomyces lanuginosa, Lipozyme RM IM from
Rhizomucor miehei, and Novozym 435 from Candida
antarctica, was determined in acidolysis conducted in
hexane. Tripalmitin with a mixture of equimolar quantities
of C18 UFAs was used as the substrate. Significantly dif-
ferent incorporation rates were observed for C18 UFAs
used (p \ 0.05). The highest incorporation was obtained
for all three C18 UFAs with Novozym 435 followed by
Lipozyme RM IM and Lipozyme TL IM catalyzed acid-
olysis under default conditions (substrate mole ratio 1:1;
temperature 50 C; reaction time 6 h; enzyme dosage
10%). Incorporation of the equimolar quantities of C18
UFAs was in the order C18:3 [ C18:2 [ C18:1 which also
reflects C18 UFAs preferences of the lipases. The effects of
operating variables on incorporation or UFA selectivity of
lipases were also investigated. Among the experimental
parameters including the mole ratio of fatty acid to triolein,
temperature, enzyme dosage, and time on incorporation,
the effect of the substrate mole ratio on UFA selectivity
was greater than those of the others.
Keywords Lipase  Selectivity  Acidolysis 
Unsaturated fatty acid  Tripalmitin 
Thermomyces lanuginosa  Rhizomucor miehei 
Candida antarctica
Introduction
Lipases [triacylglycerol (TAG) acylhydrolases, E.C. 3.1.1.3]
are very versatile enzymes that catalyze a large number of
reactions. Lipases can be used as biocatalyst for hydrolysis,
esterification, acidolysis, interesterification and modification
of fats and oils [1]. The acidolysis activity of the lipases
has been widely used for transesterification between
TAG and fatty acid (FA) to produce structured lipids
(SLs) [2].
Lipases have various degrees of selectivity towards FAs
involved in fat and oil modification. Lipase specificity may
be due to the structural features of the substrate, such as FA
chain length, unsaturation, stereochemistry, physicochem-
ical factors at the interface, and differences in the binding
site of the enzyme. The reactivity of FAs may vary
depending on the composition of substrates, water activity,
nature of solvents and source of lipase [3]. There are
numerous reports in the literature evaluating the selectivity
of commercially available lipases in hydrolysis, esterifi-
cation and transesterification reactions. The FA selectivity
of commercially available lipases evaluated in acidolysis
with substrate combinations of different acyl donors and
the same TAG [4] or the same FA (acyl donor) and dif-
ferent TAGs [5]. Hamam and Shahidi [6, 7] examined the
effect of chain length, number of double bonds, the loca-
tion and geometry of double bonds, the reaction conditions,
and the reactivity of five lipases on the incorporation of
long-chain fatty acids (LCFAs) into TAGs, such as tri-
stearin, trilinolein and trilinolenin. Shimada et al. [8]
determined FA specificity of Rhizopus delemar lipase in
acidolysis using randomly interesterified oil. FA selectivity
of lipase from Geotrichum candidum was determined
in esterification reaction using LCFAs and 1-butanol by
Sonnet et al. [9].
I. Karabulut (&)  G. Durmaz  A. A. Hayaloglu
Department of Food Engineering, Inonu University,
44280 Malatya, Turkey
e-mail: ikarabulut@inonu.edu.tr
123
J Am Oil Chem Soc (2010) 87:1301–1307
DOI 10.1007/s11746-010-1613-y

Here we report an evaluation of the C18 unsaturated
fatty acid (UFA) selectivity of the lipases in acidolysis
between tripalmitin and UFA including oleic, linoleic and
linolenic acids. In addition to selectivity of the lipases, the
information given here will be useful for studies on the
synthesis of human milk fat substitute (HMFS) by lipase
catalyzed acidolysis. In general, human milk fat contains
20–25% of palmitic acid, and about 70% of that esterified
to the sn-2 position of the glycerol backbone, and the sn-1
and sn-3 positions are mostly occupied by UFAs such as
oleic acid [15]. Thus, the fats and oils containing abun-
dantly the TAG structure of tripalmitin [10–12] and/or
tripalmitin alone [13, 14] have been used to synthesize the
HMFS by acidolysis with UFAs using lipase, and clarifi-
cation of C18 UFA selectivity of lipase is required.
The purpose of the present work was to investigate C18
UFA selectivities of the commercially lipases, namely,
Lipozyme TL IM from Thermomyces lanuginosa
(immobilized on granulated silica particles), Lipozyme
RM IM from Rhizomucor miehei (immobilized on anion
exchange resin particles), and Novozym 435 from
Candida antarctica lipase B (immobilized on macroporous
acrylic resin beads) in acidolysis between tripalmitin, and
oleic, linoleic and linolenic acids. In order to emphasize the
incorporation attitude of the lipases, different reaction
conditions, effect of operating variables such as mole ratio
of C18 UFA to tripalmitin, temperature, enzyme dosage
and reaction time on incorporation was also investigated.
Materials and Methods
Materials
Lipases were provided by Novo Nordisk A/S (Bagsvaerd,
Denmark). Oleic (C18:1, cis-9-octadecenoic acid, catalog
number: O1008, 99%), linoleic (C18:2, cis,cis-9,12-octade-
cadienoic acid, catalog number: L1376, 99%), linolenic
(C18:3, cis,cis,cis-9,12,15-octadecatrienoic acid; a-linolenic
acid, catalog number: L2376, 99%) acids and tripalmitin
(catalog number: T5888, 99%) were purchased from Sigma-
Aldrich Chemie GmbH (Steinheim, Germany). Fatty acid
methyl ester (FAME) mixture (37 component FAME mix)
and mono-, di- and triglyceride mixtures were obtained from
Supelco (Bellefonte, PA). Sodium sulfate (anhydrous) was
supplied by J.T. Baker (Deventer, Holland). All other
chemicals and reagents for the analysis were analytic or
chromatography grades.
Acidolysis Reaction
Experimental design was similar to those reported in a
previous study [4]. Equimolar quantities of C18 UFAs
(C18:1 ? C18:2 ? C18:3, a total of 0.12 mmol) were
combined in hexane. The mixture of tripalmitin with a
volumetric amount of C18 UFAs solution was used in
acidolysis. Reactions were carried out in tightly closed,
screw-capped glass vials (20 mL) containing C18 UFAs
solution-tripalmitin mixture. The total volume of the
reaction mixture was 3 mL. The vials were incubated in a
shaking water bath at 200 rpm. The effects of substrate
mole ratios (C18 UFAs: tripalmitin) ranging from 1:1 to
4:1, temperatures ranging from 40 to 60 C, reaction times
ranging from 3 to 24 h, and enzyme dosages ranging from
5 to 20% (by total weight of substrates) on the incorpora-
tions were studied. To determine the effects of different
parameters on the incorporation of FAs into triolein, the
default conditions were chosen as the following: substrate
mole ratio 1:1, temperature 50 C, reaction time 6 h,
enzyme dosage 10%, and no extra water addition.
At the end of the reaction the suspensions were filtered
through syringe membrane filter (0.45 lm) to remove the
enzyme particles and filtrates (hexane solutions) were used
for subsequent analysis.
Analysis of Product
One hundred microliters of the hexane solution was applied
to thin-layer chromatography (TLC) plates (20 cm 9
20 cm) coated with silica gel 60 F254 (Merck) in a thin
uniform line by means of an applicator (Linomat 5, Camag,
Muttenz, Switzerland). The developing solvent was hex-
ane/diethyl ether/acetic acid (80:20:1, v/v/v). The bands
were visualized under UV light after spraying with 0.2%
2,7-dichlorofluorescein in ethanol. The TAG band was
scraped off into a screw-capped vial and methylated with
3 mL of 6% HCl in methanol at 75 C for 2 h [16]. At the
end of the incubation, vials were cooled on ice bags, and
2 mL of hexane was added before centrifugation. The
upper phase containing FAMEs was transferred to a vial
containing anhydrous sodium sulfate by Pasteur pipet and
used for FA composition analysis.
FA Composition Analysis
The FAMEs were analyzed by gas–liquid chromatography.
The gas chromatograph (GC) was an Agilent 7890A with a
fused capillary column (DB-23, 60 m 9 0.25 mm i.d.,
0.25 lm film thickness; J&W Scientific, Folsom, CA), an
auto injector (Agilent 7683B), and a flame ionization
detector (FID) and was operated in split mode with the split
ratio of 1:30. The injector and detector temperatures were
maintained at 250 C. The column temperature was held
at 140 C for 5 min and ramped to 240 C for 10 min at
the rate of 4 C per min. The carrier gas was helium, and
the total flow rate was 30 mL/min. The FAMEs were
1302 J Am Oil Chem Soc (2010) 87:1301–1307
123