Lipase activity and enantioselectivity of whole cells from a wild-type Aspergillius flavus strain

Article abstract of DOI:10.1016/j.molcatb.2013.12.005

This study reports the high enantiomeric preference of whole cell lipase from Aspergillus flavus wild-type that allows the preparation of a chiral secondary alcohol. Whole cells prepared from a wild-type Aspergillus flavus strain were used as biocatalysts to prepare (R)-1-phenylethyl acetate. (R)-1-Phenylethanol was esterified into (R)-1-phenylethyl acetate with a 94.6% enantiomeric excess (ee) within 24 h at 40 C and (S)-1-phenylethanol remained in the reaction medium with a >99%ee. Besides, this biocatalyst allows the preparation of ethyl laurate and a mixture of 2-chloro-1-(chloromethyl)ethyl acrylate and 2,3-dichloro-1-propyl acrylate. The ethyl laurate yield was 96%, whereas the synthesis of a mixture of the acrylate regioisomers, 2-chloro-1-(chloromethyl)ethyl acrylate and 2,3-dichloro-1-propyl acrylate gave similar yields to those obtained using commercial lipases.

Full text of DOI:10.1016/j.molcatb.2013.12.005

Journal of Molecular Catalysis B: Enzymatic 100 (2014) 78–83
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Lipase activity and enantioselectivity of whole cells from a wild-type
Aspergillius flavus strain
Carmen Solartea,1, Edinson Yara-Varón , Jordi Eras , Mercè Torres , Mercè Balcells ,
Ramon Canela-Garayoa
a
a,1
a
b
a
a,∗
Department of Chemistry, University of Lleida, Rovira Roure 191, Lleida 25198, Spain
Department of Food Science and Technology, University of Lleida, Rovira Roure 191, Lleida 25198, Spain
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 July 2013
Received in revised form
This study reports the high enantiomeric preference of whole cell lipase from Aspergillus flavus
wild-type that allows the preparation of a chiral secondary alcohol. Whole cells prepared from a
wild-type Aspergillus flavus strain were used as biocatalysts to prepare (R)-1-phenylethyl acetate. (R)-
1
8 November 2013
1
-Phenylethanol was esterified into (R)-1-phenylethyl acetate with a 94.6% enantiomeric excess (ee)
Accepted 9 December 2013
Available online 17 December 2013
◦
within 24 h at 40 C and (S)-1-phenylethanol remained in the reaction medium with a >99%ee. Besides,
this biocatalyst allows the preparation of ethyl laurate and a mixture of 2-chloro-1-(chloromethyl)ethyl
acrylate and 2,3-dichloro-1-propyl acrylate. The ethyl laurate yield was 96%, whereas the synthesis of a
mixture of the acrylate regioisomers, 2-chloro-1-(chloromethyl)ethyl acrylate and 2,3-dichloro-1-propyl
acrylate gave similar yields to those obtained using commercial lipases.
Keywords:
Aspergillus flavus
(R)-1-Phenylethyl acetate
Ethyl laurate
© 2013 Elsevier B.V. All rights reserved.
2
2
-Chloro-1-(chloromethyl)ethyl acrylate
,3-Dichloro-1-propyl acrylate
1
. Introduction
Recent years have witnessed the rapid development of lipase
poly(dichloropropyl acrylates), which have applications in many
fields [7].
Lipase-catalyzed biotransformation is one of the most popular
and practical enzymatic technologies. Lipases (triacylglycerol acyl-
hydrolases, EC 3.1.1.3) are enzymes that catalyze a broad spectrum
of reactions, such as the hydrolysis of ester bonds and transes-
terification and ester synthesis, at the interface between substrate
and water or in non-aqueous organic solvents [8]. Consequently,
lipases are used in a wide range of industrial processes, includ-
ing food, chemical, pharmaceutical and detergent production. In
many cases, however, these enzymes have been used in an immo-
bilized form, which is costly and time-consuming, thus hindering
the widespread use of enzymatic processes. Lipases from various
microorganisms have been reported. There are two types of lipase
preparation: (i) extracellular lipase, which is secreted into culture
broth and (ii) intracellular or whole cell lipase, which remains
either inside the cell or in the cell-wall [9]. Many of these have
been purified and their properties are described. To date, several
lipases are commercially available, and their applications have been
extensively described [10,11]. However, while most of these are
extracellular enzymes, little research has been devoted to intracel-
lular or cell-bound lipases.
applications for the preparation of esters and chiral alcohols. This
growth, in part, is driven by EU and US legislation stating that
natural flavouring and fragrance products can be prepared from
natural sources only by physical processes (extraction from nat-
ural sources) or by biotechnological transformations that involve
precursors isolated from nature [1]. Indeed, several biotransfor-
mation processes using either enzymes or whole cells have been
implemented to conduct the kinetic resolution of 1-phenylethanol
a compound used in various pharmaceutical and personal care
products [2–5].
Biocatalysts have also been reported for the preparation of ethyl
laurate, which has a coconut aroma [6]. A regioisomeric mixture
of 2-chloro-1-(chloromethyl)ethyl acrylate and 2,3-dichloro-1-
propyl acrylate (DCPA), have been used as monomers to prepare
Abbreviations: aw, water activity; PDA, potato/dextrose/agar; r, specific
activity; R-1-PEA, (R)-1-phenylethyl acetate; ees, enantiomeric excess substrate;
eep, enantiomeric excess product; DCPA, mixture of regioisomers (2-chloro-1-
(
chloromethyl)ethyl acrylate and 2,3-dichloro-1-propyl acrylate); CALB, lipase B
Cell-bound lipases are economically attractive because they can
be produced at low cost. The biomass can be used directly, thus
avoiding isolation, purification and immobilization procedures.
Moreover, the biocatalyst can be easily recovered by filtration. Also,
from Candida antarctica.
∗ Corresponding author. Tel.: +34 973 702843; fax: +34 973238264.
E-mail address: canela@quimica.udl.cat (R. Canela-Garayoa).
These authors contributed equally to this paper.
1
1
381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.12.005

C. Solarte et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 78–83
79
naturally immobilized lipases show potential for applications. Nagy
2.3. Preparation of whole cell lipase
et al. [3], studied thirty-eight filamentous fungi cultivated under
solid state fermentation (SSF). The majority of these preparations
proved to be effective as enantiomer selective biocatalyst and some
were successfully applied in preparative scale kinetic resolution of
secondary alcohols.
Mycelium obtained from the culture medium was harvested
using a Buchner funnel, washed with distilled water followed by
acetone, and freeze-dried for 18 h. It was then ground to powder
consistency.
The use of naturally immobilized lipases has been proposed
in the oil and fat industry [12,13], in the preparation of flavours
2.4. Equilibration of water activity (aw)
[
1,14] and, recently, in the synthesis of biodiesel [15,16]. Recom-
binant microbial whole-cell biocatalysts expressing lipases have
also been proposed for enantioselective transesterification in non-
aqueous medium. Nevertheless, the increasing concern regarding
non-natural approaches for the preparation of additives and fra-
grances has prompted us to look for wild microorganisms that could
be used to prepare aroma and fragrance compounds.
In our attempt to obtain a new fungal cell biocatalyst from our
environment, we isolated a lipolytic Aspergillus flavus strain from
sunflower seeds collected in a sunflower mill. Several studies under
submerged fermentation and solid-state fermentation has been
performed to produce whole cell lipase from A. flavus. Its stability
and activity has been reported [17–21]. Here we describe several
characteristics of the whole cell lipase prepared from the isolated
wild-type A. flavus strain. These whole cells were used as biocata-
lysts to prepare (R)-1-phenylethyl acetate, ethyl laurate and DCPA.
In this study we describe the high enantiomeric preference of a
wild-type whole cell lipase from A. flavus that allows the prepara-
tion of a chiral secondary alcohol.
The aw in the experiments was set by independently equili-
brating reagents, solvent and biocatalyst with aqueous saturated
solutions of LiCl (aw = 0.12), MgCl2 (aw = 0.33), K CO3 (aw = 0.42),
2
Mg(NO ) (aw = 0.54) and NaCl (aw = 0.75). Separate closed contain-
3
2
ers were used for each reactant and biocatalyst [22]. Equilibration
was performed at room temperature for at least 48 h. The aw of
the biocatalyst was measured using an Aqua Lab series 3TE from
Decagon Devices Inc. (Pullman, WA, USA).
2
.5. Biocatalyst activity
Two mL of a 0.09 M isooctane solution of oleic acid (0.05 g)
◦
containing 0.08 g of 1-propanol was stirred for 1 h at 28 C in the
presence of various amounts of whole cell lipase (aw = 0.12). Sam-
ples were collected every 15 min. These samples were diluted at
−1
ca. 1 mg mL using methyl palmitate as internal standard. Result-
ing samples were analyzed by gas-chromatography (GC-FID) as
described below. All experiments were performed in triplicate.
2
. Materials and methods
2.6. Evaluation of substrate adsorption by the whole cell lipase
biomass
A strain of Aspergillus flavus was isolated from sunflower seeds
◦
and was maintained on potato/dextrose/agar (PDA) at 4 C. The
microorganism was deposited in the culture collection “Colección
Espa n˜ ola de Cultivos Tipo” (Burjassot, Valencia-Spain), reference
number CECT 20475.
Eighty mL of a 0.022 M isooctane solution of methyl oleate
(500 mg) containing 3 g of whole cell lipase (aw = 0.12) were stirred
◦
for 24 h at 28 C. Samples were collected at 0, 2, 4 and 24 h. These
−
1
samples were diluted at ca. 1 mg mL using methyl palmitate as
internal standard and analyzed by gas chromatography (GC-FID) as
described below. All experiments were performed in triplicate.
2.1. Reagents and solvents
2.7. Kinetic resolution of rac-1-phenylethanol
Asparagine, K HPO , MgSO , glucose, thiamine hydrochlo-
2
4
4
ride, Fe(NO ) .9H O, ZnSO ·7H O, isopropenyl acetate, rac-1-
3
3
2
4
2
Kinetic resolution reactions were carried out using dry condi-
tions in flame-dried glassware following a previously described
phenylethanol, (S)-1-phenylethanol, lauric acid, and toluene were
purchased from Sigma–Aldrich (Sigma–Aldrich Quimica, S.A.,
Madrid, Spain). MnSO ·H O was supplied by Fisher Scientific
method [23]. rac-1-Phenylethanol was dried over a molecular sieve
4
2
(
4 A˚ ) before use. Isopropenyl acetate was dried over CaCl and dis-
2
(
Madrid, Spain). Oleic acid was acquired from Merck (Barcelona,
tilled before use. Dry toluene was dried by refluxing under nitrogen
in the presence of sodium wire and benzophenone.
Whole cell lipase (25 mg) and sodium carbonate (53 mg,
.5 mmol) were added to a vial. The corresponding rac-1-
Spain). Ethyl laurate, ethyl acrylate, methyl palmitate and methyl
oleate were from Fluka (Sigma–Aldrich, Madrid, Spain). Ethanol
and hexane were supplied by J.T. Baker (Quimega, Lleida, Spain).
Isooctane and sodium carbonate were purchased from Panreac
0
phenylethanol or (S)-1-phenylethanol (55 mg, 0.5 mmol) dissolved
in dry toluene (1 mL) was added to the vial, and the mixture was
stirred for 6 min. Next, isopropenyl acetate (110 ␮L, 1.0 mmol) was
added to the reaction. Experiments were carried out at 25, 40 and
(
Barcelona, Spain). 1-Propanol was from Acros Organics (Barcelona,
Spain).
◦
2.2. Microorganism, growth media, and culture conditions
60 C. Samples were collected between 30 min and 44 h depending
on the temperature and reagent used, and then analyzed by chiral
GC as described below. A blank study was conducted without using
isopropenyl acetate.
A non-aflatoxigenic strain of A. flavus, isolated from sunflower
seeds, was cultivated in a synthetic liquid medium that con-
tained 2.0 g asparagine, 1.0 g K HPO , 0.5 g MgSO , 5.0 mg thiamine
2
4
4
hydrochloride, 1.45 mg Fe(NO ) ·9H O, 0.88 mg ZnSO ·7H O and
3
3
2
4
2
2.8. Preparation of ethyl laurate
0
.31 mg MnSO ·H O per litre of distilled water. The pH was
4
2
adjusted to 6.0 using 1 M HCl. Two hundred and fifty millilitres
of the liquid medium were sterilized in a 1 L Erlenmeyer flask
−1
−1
Half
a
mL of
a
solution containing 25 mg mL
−1
(
(
0.125 mmol mL ) of lauric acid and ethanol 11.52 mg mL
0.250 mmol mL ) in hexane was added to a reaction vial (1.5 mL)
◦
at 121 C for 15 min, and 2% of refined sunflower oil was added.
−1
The medium was inoculated with 2.5 mL of a spore suspension
fitted with a PTFE-lined cap. Then 20 mg of biocatalyst (aw = 0.54)
6
(
5 × 10 spores/mL) of A. flavus grown on PDA. The medium was
was added to the vial, and the mixture was stirred and heated to
◦
then incubated at 28 C for 5 days on an orbital shaker at 200 rpm.
◦
4
0 C for 24 h. The resulting solution was analyzed using GC-FID.

8
0
C. Solarte et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 78–83
Product quantification was achieved using a standard curve
obtained from ethyl laurate.
100
80
60
40
20
0
2
.9. Procedure for the preparation of 2,3-dichloro-1-propyl
laurate
2
-Chloro-1-(chloromethyl)ethyl laurate was prepared from
glycerol and lauric acid following a previously described method
[
24]. The isomerization of 2-chloro-1-(chloromethyl)ethyl lau-
rate to 2,3-dichloro-1-propyl laurate was achieved by microwave
irradiation (300 W max, 17 atm max, 243–247 C) for 1 h in a
◦
2.50
1.25
0.63
0.33
0.17
Substrate:whole cell lipase ratio (w/w)
solvent-free system. The isomerization process used was previ-
ously evaluated [25].
Fig. 1. Effect of the substrate:whole cell lipase ratio on the specific activity of A.
flavus, calculated on the basis of the synthesis of propyl oleate from oleic acid and
2.10. Enzymatic transesterification
1-propanol.
A 1:1 mixture of ethyl acrylate (1 mmol, 100 mg) and dichloro-
specific activity (r) of the whole cell lipase. This specific activity
propyl laurates (32:68 isomeric mixtures, 1 mmol, 311 mg) was
was determined at five substrate:whole cell lipase ratios (Fig. 1).
®
continuously reciprocally shaken (Eppendorf Thermomixer Com-
−1 −1
, a range that
The activity was between 65 and 91 ␮mol min
g
fort) at 1200 rpm at atmospheric pressure in a reaction vial (1.5 mL)
fitted with a PTFE-lined cap. Reaction vials were used as received.
could be a consequence of the inherent heterogeneity of the cells.
No apparent ratio trends were observed in the total number of
reactions performed. Nevertheless, the standard deviation for a
given ratio did not exceed 7% (ratio 2.50).
Considering that the fungal biomass used as biocatalyst could
partially retain the product and the reagent used, a new set of
experiments was conducted to preclude this possibility. Fig. 2
shows that the average percentage of methyl oleate absorbed by
A set of experiments were carried out in a solvent-free system at
◦
4
0 C using 15% (47 mg) of biocatalyst on the basis of the weight of
dichloropropyl laurates. Samples were collected at 24 h. Once the
experiment had ended, an aliquot of 10 mg of the crude product was
dissolved in hexane containing butyl acrylate as internal standard.
The resulting solution was analyzed by GC-FID. Each compound
was quantified using the internal standard. Experiments were per-
formed in triplicate.
1
g of whole cell lipase after 24 h using a substrate:whole cell lipase
ratio of 0.12 was 5%. Given these results obtained with the lowest
substrate:whole cell lipase ratio, we performed all the subsequent
experiments using substrate:whole cell lipase ratios higher than
2
.11. GC analysis
1
.7 to assure that the biocatalyst could not retain any chemical
The progress of each reaction was determined by GC using
compound.
an Agilent (Barcelona, Spain) HP6890 series gas chromatograph
coupled to an FID detector. A 30 m × 0.25 mm fused silica capil-
lary coated with a 0.20-␮m film of poly(80% biscyanopropyl-20%
cyanopropylphenyl siloxane) (SP-2330; Supelco, Madrid, Spain)
was used for fatty acid analysis.
3.1. Kinetic resolution of rac-1-phenylethanol using fungal whole
cell lipase
Table 1 shows the progress of the kinetic resolution of rac-1-
◦
The temperature programme used was 40 C for 5 min, followed
phenylethanol (1) at three temperatures. Whole cell lipase from A.
flavus were used at a substrate:whole cell lipase ratio of 2.0 and
aw = 0.12 (Fig. 3A). The conversion to (R)-1-phenylethyl acetate (3)
◦
by an increase of 20 C/min until reaching the final temperature of
◦
2
25 C, which was then held for 3 min. A 1:20 split injection ratio
was applied. Hydrogen was used as the carrier gas at a constant
(R-1-PEA) calculated as ees/(ees + eep) was 48% with 96%ee (E = 150)
pressure of 620 kPa. The injection volume was 1 ␮L. The injection
◦
within 44 h of reaction at 25 C with an apparent 87% ees. Never-
theless, the conversion reached 51% with 94.6%ee (E > 200) within
2
showed >99% ees. The increase in the reaction temperature (60 C)
did not lead to an improvement of these results. The conver-
sion rate was 49.5% for 12 h of reaction at this temperature and
R-1-PEA dropped to 92.2%ee (E = 73). An experiment was carried
out using (S)-1-phenylethanol. The formation of 1.56% of (S)-1-
phenylethanol acetate (4) was observed after 24 h of reaction at
◦
◦
system was held at 250 C and the FID system at 280 C. Quantifica-
tion was carried out by a conventional internal standard calibration
method using the corresponding ester standard.
◦
4 h of reaction at 40 C. In this case, the remaining substrate
◦
Chiral analysis was performed using a 25 m × 0.250 mm fused
silica capillary coated with a 0.25-␮m film of modified ␤-
cyclodextrins bonded to dimethylpolysiloxane (CP-Chirasil-Dex CB
Varian, Madrid, Spain). The oven temperature was held constant at
◦
1
20 C for 10 min. A 100:1 split injection ratio was applied. Hydro-
gen was used as the carrier gas at flow rate of 2.0 mL/min. The
◦
injection volume was 1 ␮L. The injection system was held at 250 C
and the FID system at 250 C.
◦
180
1
1
50
20
3
. Results and discussion
90
Several studies have been carried out to describe the optimal
conditions for the production of lipase by A. flavus [17–19]. In
our research group we have used an optimal method to cultivate
the fungal mycelium [26]. In this method, refined sunflower oil
was added to induce the lipase activity. Mycelium was harvested
from the culture medium, washed with water and acetone,
60
30
0
0
2
4
24
Time (h)
−
1
freeze-dried and ground to a powder. The yield was 10 g L
.
Finally, the mycelium was equilibrated at various aw. An initial
study was carried out using pure oleic acid to investigate the
Fig. 2. Effect of contact time on the absorption of methyl oleate (FAME) by the whole
cell lipase from A. flavus. The substrate:whole cell lipase ratio used was 0.12.

C. Solarte et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 78–83
81
Table 1
Time course of (R)-1-phenylethyl acetate (R-1-PEA) yield in enantioselective transesterification at various temperatures using whole cell lipase from a wild-type A. flavus
strain.
◦
◦
◦
T = 60 C
t (h)
T = 25
C
T = 40
C
ees (%)a
eep (%)a
c (%)b
Ec
ees (%)a
eep (%)a
c (%)b
Ec
ees (%)a
eep (%)a
c (%)b
Ec
0
1
2
3
5
7
9
1
2
4
.5
0
0
2
–
–
–
–
–
46
87.1
0
0
>99
–
–
–
0
0
2.2
–
–
–
–
–
31.7
47.5
–
–
>200
<1
1.2
2.7
>99
>99
>99
>99
>99
98.4
97.0
95.0
94.6
–
<1
>200
>200
>200
>200
>200
166
101
69
1.6
3.7
>99
>99
>99
>99
>99
>99
94.2
92.2
–
1.5
3.9
9.2
15.2
16.7
27.0
43.9
49.5
–
>200
>200
>200
>200
>200
>200
71
73
–
–
1.2
2.7
5.0
9.7
22.3
30.6
36.9
51.4
–
10.2
17.9
19.5
36.9
73.7
90.3
–
–
–
–
–
–
4.8
10.7
28.2
42.7
55.6
>99
–
–
–
2
4
4
>99
96.2
>200
150
>200
–
–
–
–
a
b
c
Determined by chiral GC.
c = ees/(ees + eep).
E = ln[(1 − c) × (1 + eep)]/ln[(1 − c) × (1 − eep)].
◦
4
0 C, thereby confirming the high enantiopreference of whole cell
to selectively synthesize R-1-PEA from rac-1-phenylethanol and
lipase from A. flavus for (R)-1-phenylethanol.
vinyl acetate. The yield and enantiomeric excess (%ee) of R-1-PEA
after 48 h of reaction at 30 C using a substrate:resting cell ratio
◦
Several studies have addressed the enantioselective activity of
intracellular enzymes present in fungal cells. Thus, a recombi-
nant strain overexpressing Aspergillus oryzae lipase derived from
the wild-type strain RIB40 was immobilized in biomass-support
particles and used as a whole-cell biocatalyst. The immobilized
lipase-overexpressing strain showed high activity and was used
of 0.4 was 90% and 95%ee, respectively [27]. The same A. oryzae
whole-cell biocatalyst expressing the lipase-encoding gene from C.
antarctica shows a higher performance in the enantiomeric transes-
terification of rac-1-phenylethanol, yielding 88.1% of R-1-PEA with
a 99.9%ee after 3.5 h of reaction [19,28]. Although this performance
Fig. 3. Biocatalytic reactions using whole cell lipase from an A. flavus strain. (A) Kinetic resolution of rac-1-phenylethanol. (B) Synthesis of ethyl laurate. (C) Synthesis of
mixture of regioisomers (DCPA).

8
2
C. Solarte et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 78–83
is superior to that of the A. flavus resting cell, it should be con-
sidered that it was reached by expressing C. antarctica lipase, the
enzyme shown to provide the highest yield and ee [29], and using
a substrate:whole-cell ratio of 0.6. This ratio implies that those
authors used approximately three-fold more biocatalyst than that
used in the present study in the same reaction with wild resting
cells. Nagy et al. [3] studied 38 filamentous fungi cultivated under
SSF conditions for kinetic resolutions of racemic secondary alco-
hols. The freeze-dried solid prepared form two Aspergillus species
flexibility and activity of the lipase. These dichloropropyl esters can
be readily transformed into polymeric compounds [7].
4. Conclusions
The kinetic resolution of racemic 1-phenylethanol was suc-
cessfully achieved using whole cell lipase from a wild-type A.
flavus strain. The (R)-1-phenylethanol was transesterified into
(R)-1-phenylethyl acetate, leaving the (S)-1-phenylethanol in the
(
Aspergillus terreus and Aspergillus niger) were tested. The reac-
reaction medium with >99%ee. Our results show that the biocat-
alyst proved highly selective, yielding (R)-1-phenylethyl acetate
tion was conducted for 120 h at room temperature. The acetylation
of racemic 1-phenylethanol showed a conversion of 45.6% and
◦
in 24 h at 40 C with 94.6%ee. Moreover, we have shown that this
8
2
7.8%ee of R-1-PEA by A. terreus. A. niger showed a conversion of
4% and 56.9%ee of R-1-PEA. Both biocatalysts showed lower enan-
biocatalyst shows acceptable performance in esterification and
transesterification reactions. Ethyl laurate can be prepared at very
high yield while the regioisomeric mixture of DCPA can be syn-
thesized at similar yields to those obtained using a commercial
lipase.
tiomeric excess of R-1-PEA than that showed by our strain of A.
flavus. The best biocatalyst in terms of ee (98.4%) described by these
authors was the solid material prepared from a Gliocladium catenu-
latum strain. Nevertheless, they used a substrate:biocatalyst ratio
of 1 in all the studies, twice than that used in our study.
Acknowledgements
3
.2. Biocatalytic synthesis of ethyl laurate
This work was supported in part by a Grant-in-Aid for the
Secretaría de Estado de Política Científica y Tecnológica of the Span-
ish Ministry of Education and Culture (contract grant number:
CTQ2009-14699-C02-01). The authors are grateful to the Comis-
sionat per a Universitats i Recerca del Departament d’Innovació,
Universitats i Empresa de la Generalitat de Catalunya and to the
European social Fund (EFS) for the FI grant of Carmen Solarte Orozco
and to the Vicerrectorat de Recerca de l’Universitat de Lleida for the
UdL grant of Edinson Yara Varón. We also thank Tania Yates for the
proofreading of the manuscript.
The biocatalytic esterification of lauric acid (5) was carried out
using ethanol (6) and the whole cell lipase from A. flavus (Fig. 3B).
Ethyl laurate (7) was synthesized with a 95% yield after 24 h of
reaction. This result was similar to that described by [6], who
®
reported a 92% yield of ethyl laurate using Lipolase , a commercial
preparation containing the lipase from Thermomyces lanuginosa.
Mycelium-bound lipase of a locally isolated A. flavus have been
used previously to catalyze the acidolysis of several vegetables oils
[
21] and the transesterification of acylglycerides to the production
of FAME [30]. The works mentioned above demonstrate that A.
flavus have been identified as robust whole-cell biocatalysts. The
use of lipases as catalysts allows the preparation of high purity
esters. Moreover, the products prepared using these enzymes can
be labelled “natural”, provided that natural raw materials are also
used.
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