Specific enzyme-catalyzed hydrolysis and synthesis in aqueous and organic medium using biocatalysts with lipase activity from Aspergillus niger MYA 135

Article abstract of DOI:10.1007/s10562-012-0901-6

In the present study, the specific hydrolytic activity of three biocatalysts such as the constitutive mycelium-bound lipase, the induced mycelium-bound lipase and the lyophilized induced supernatant from A. niger MYA 135 was evaluated in both aqueous and organic media.A direct correlation between activity in water and n-hexane was not observed for the same hydrolytic reaction. The n-hexane/water activity ratio (R_O/A) was applied to characterize the activity in organic medium. The three biocatalysts showed R_O/A values higher than 1 for hydrolysis of long-chain fatty acid esters, demonstrating a higher specific hydrolytic activity in organic solvent than in water. A different behavior was observed during hydrolysis of middle-chain fatty acid esters, which was higher in aqueous medium (R _O/A<1). Transesterifications of different alcohols with various p-nitrophenyl derivatives using all three biocatalysts preparations were also evaluated in nhexane. For methanolysis and ethanolysis, the constitutive mycelium-bound lipase displayed an interesting preference for C16 substrate (p-nitrophenyl palmitate). The induced mycelium-bound lipase showed high specific transesterification activities in the presence of water-miscible alcohols and middle-chain fatty acid esters (p-nitrophenyl caprate and p-nitrophenyl laurate), being the highest specific transesterification activity (91.4 ± 1.7 mU/g_dw) observed in a reaction mixture containing propanol and p-nitrophenyl laurate. Finally, both p-nitrophenyl caprate (C10) and p-nitrophenyl laurate (C12) were preferentially methanolized by the lyophilized induced supernatant, being this lipase activity the most specific biocatalyst preparation under transesterification conditions. A selectivity-based analysis of each lipase preparation toward transesterification or hydrolysis in organic medium was evaluated as well. Springer Science+Business Media, LLC 2012.

Full text of DOI:10.1007/s10562-012-0901-6

Catal Lett (2012) 142:1361–1368
DOI 10.1007/s10562-012-0901-6
Specific Enzyme-Catalyzed Hydrolysis and Synthesis in Aqueous
and Organic Medium Using Biocatalysts with Lipase Activity
from Aspergillus niger MYA 135
Cintia M. Romero
•
Licia M. Pera
•
Flavia Loto Mario D. Baigori
•
Received: 31 May 2012 / Accepted: 26 August 2012 / Published online: 20 September 2012
Ó Springer Science+Business Media, LLC 2012
Abstract In the present study, the specific hydrolytic
activity of three biocatalysts such as the constitutive
mycelium-bound lipase, the induced mycelium-bound
lipase and the lyophilized induced supernatant from
A. niger MYA 135 was evaluated in both aqueous and
organic media. A direct correlation between activity in water
and n-hexane was not observed for the same hydrolytic
reaction. The n-hexane/water activity ratio (RO/A) was
applied to characterize the activity in organic medium. The
three biocatalysts showed RO/A values higher than 1 for
hydrolysis of long-chain fatty acid esters, demonstrating a
higher specific hydrolytic activity in organic solvent than in
water. A different behavior was observed during hydrolysis
of middle-chain fatty acid esters, which was higher in
aqueous medium (RO/A \ 1). Transesterifications of differ-
ent alcohols with various p-nitrophenyl derivatives using all
three biocatalysts preparations were also evaluated in n-
hexane. For methanolysis and ethanolysis, the constitutive
mycelium-bound lipase displayed an interesting preference
for C16 substrate (p-nitrophenyl palmitate). The induced
mycelium-bound lipase showed high specific transesterifi-
cation activities in the presence of water-miscible alcohols
and middle-chain fatty acid esters (p-nitrophenyl caprate and
p-nitrophenyl laurate), being the highest specific transeste-
lyophilized induced supernatant, being this lipase activity
the most specific biocatalyst preparation under transesteri-
fication conditions. A selectivity-based analysis of each
lipase preparation toward transesterification or hydrolysis in
organic medium was evaluated as well.
Keywords Lipase ꢀ Aspergillus niger ꢀ Hydrolysis ꢀ
Transesterification ꢀ Organic medium ꢀ Aqueous medium ꢀ
Substrate specificity
1 Introduction
Among all the enzymes that show significant activity in
organic media, lipases are of particular interest because of
their commercial potential in many applications [1, 2].
Lipases have been widely used as common reagents for
ester hydrolysis and synthesis, and lipase-catalyzed reac-
tions are often carried out in organic solvents [3]. However,
organic solvents frequently alter enzymatic activity,
enzyme stability and substrate specificity [4]. Observations
in the three-dimensional structure revealed that organic
solvent stability of lipases is their intrinsic property and
unique with respect to each lipase [5].
rification activity (91.4 ± 1.7 mU/g ) observed in a reac-
dw
Guidelines have been provided to predict enzyme
activity in organic media [6]. For example, the amount of
water present in the system should be expressed in terms of
its thermodynamic activity instead of its concentration.
Halling [7] observed that a change in the reaction solvent
or in the support used for immobilization showed no effect
on the quantity of water required for maximum activity. In
addition, certain enzymes equally accelerated reactions
catalyzed in water or organic media [8]. However, very few
studies have been carried out to predict activity in organic
tion mixture containing propanol and p-nitrophenyl laurate.
Finally, both p-nitrophenyl caprate (C10) and p-nitrophenyl
laurate (C12) were preferentially methanolized by the
C. M. Romero ꢀ L. M. Pera ꢀ F. Loto ꢀ M. D. Baigori (&)
Planta Piloto de Procesos Industriales Microbiol o´ gicos
(
4
PROIMI)-CONICET, Av. Belgrano y Pasaje Caseros,
000 Tucum a´ n, Argentina
e-mail: lymb32@gmail.com
0
solvents compared to that in water. Pencreac h and Baratti
123

1
362
C. M. Romero et al.
[
9] proved that activity in organic media could not be
2.3 Enzyme Production
estimated from that in water.
Lipase specificity has been studied extensively. In most
cases, different kinds of specificity or selectivity can be
distinguished during hydrolysis or synthesis reactions [10].
Chain-length specificity has been related to structural fea-
tures of lipases [11, 12], but to date no lipase has been
found to be strictly specific for a given chain length.
Knowledge about substrate specificity would simplify the
choice of a specific biocatalyst for a certain reaction.
Chain-length specificity of lipase could be modified when
these enzymes are immobilized [13]. Enzyme immobiliza-
tion has proven to be a useful technique to enhance enzy-
matic activity in organic media [14, 15]. Immobilization
generally stabilizes the enzyme, enhances lipase properties
such as thermostability and activity in a non-aqueous med-
ium and improves flexibility of the enzyme/substrate com-
plex [13]. There exists a variety of methods to immobilize
lipases, using many different types of support. Techniques
have roughly been classified into five categories: adsorption,
covalent binding, entrapment, microencapsulation and nat-
urally immobilized biocatalysts (whole cell biotransforma-
tion) [16]. Whole-cell biocatalysts can be readily and
cheaply cultured in large quantities, such whole-cell systems
could become promising and inexpensive biocatalysts [17].
Thus, among the established whole-cell biocatalyst systems,
filamentous fungi have arisen as the most robust whole-cell
biocatalyst for industrial applications [18].
Fermentation was carried out at 30 °C in 500-ml shake
flasks (250 rpm) containing 100 ml of fermentation med-
ium. Culture flasks were inoculated with 10 ml of a conidial
6
suspension (about 10 conidia/ml) from a stock culture.
After 24 h of incubation, the culture was transferred to
another 500-ml shake flask containing either 50 ml 2 %
(v/v) olive oil or distilled water and further incubated for
4 days under the same conditions. The mould developed a
pelleted growth form. Mycelium was collected and washed
with acetone by filtration at 4 °C for 3 min at 6,000 g; cells
were used as enzyme source. Calibration curves were
generated with wet and dry mycelium developed in medium
2
without olive oil (R = 0.973) or supplemented with 2 %
2
olive oil (R = 0.982). Supernatant obtained after filtration
of olive oil-containing medium was also used as enzyme
source. This lipase preparation was lyophilized and protein
content was determined according to Bradford [19].
2.4 Specific Hydrolytic Activity in Aqueous
and Organic Media
Specific hydrolytic activity in either aqueous or organic
medium was measured using the following substrates dis-
solved in acetone: pNPCa (p-nitrophenyl caprate), pNPL
(p-nitrophenyl laurate), pNPP (p-nitrophenyl palmitate)
and pNPS (p-nitrophenyl stearate). In aqueous solvents,
about 0.010 g of wet mycelium or lyophilized supernatant
was added to 1 ml of 100 mM phosphate buffer (pH 7.0),
containing 2 mM p-NP derivative, 0.1 % (w/v) gum Arabic
and 0.4 % (w/v) Triton X-100 [20]. The molar extinction
coefficient of p-nitrophenol (p-NP) under the given assay
In this work, the reactivity of three lipase preparations
such as the constitutive mycelium-bound lipase, the
induced mycelium-bound lipase and the lyophilized
induced supernatant obtained from Aspergillus niger MYA
1
35 was studied under hydrolytic and synthetic conditions.
-
1
-1
A selectivity-based analysis of each biocatalyst preparation
toward transesterification or hydrolysis in organic medium
was also evaluated.
conditions was 0.0103 lM cm . In organic solvents,
about 0.010 g of wet mycelium or lyophilized supernatant
was added to 1 ml of n-hexane containing 2 mM p-nitro-
phenyl derivative [21]. The reaction mixture was shaken
(150 rpm) at 37 °C. The absorbance of the supernatant
2
Experimental Section
containing p-NP was determined at 405 nm. The p-NP was
extracted with 1 ml 0.25 M Na CO from n-hexane before
measurement. The molar extinction coefficient of p-NP
2
3
2
.1 Microorganism and Maintenance
-
1
-1
under these assay conditions was 0.0205 lM cm . One
unit of enzyme activity was defined as the amount of
biocatalyst that released 1 lmol of p-NP per min. Specific
activity was expressed as milliunits per gram of dry cell
weight or protein for mycelium and lyophilized superna-
tant, respectively.
Aspergillus niger ATCC MYA 135, formerly A. niger 419
from the PROIMI culture collection, was used throughout
this work. It was maintained by monthly transfers onto
glucose–potato agar slants, incubated at 30 °C and stored at
4
°C.
2
.2 Fermentation Medium
2.5 Enzymatic Transesterification of p-Nitrophenyl
Derivatives with Different Alcohols
The fermentation medium was as follows (in g/l): sucrose,
0.0; KH PO , 1.0; NH NO , 2.0; MgSO ꢀ7H O, 2.0;
1
Enzymatic transesterification was carried out following the
2
4
4
3
4
2
CuSO , 0.06. The initial pH was adjusted to 7.0 with NaOH.
4
technique of Romero et al. [22]. Briefly, 100 ll of 20 mM
1
23

Specific Lipase-Catalyzed Hydrolysis
1363
p-nitrophenyl derivative dissolved in acetone, 100 ll of
each alcohol and about 0.010 g of wet mycelium or
lyophilized supernatant were added to 800 ll of n-hexane.
The reaction mixture was shaken (150 rpm) for 1 h at
extract was able to produce ethyl estearate in a solvent-free
system with a transesterification activity of 0.78 ± 0.01 U/l
[26].
Thus, taking in mind a future application in food,
pharmaceutical and energy industries, the specific hydro-
lytic activity of three biocatalyst systems such as the
constitutive mycelium-bound lipase, the induced mycelium-
bound lipase and the lyophilized induced lipase supernatant
from A. niger MYA 135 was determined against various
substrates in both aqueous and organic media. In addition,
the selectivity of the three biocatalysts toward transesterifi-
cation or hydrolysis reaction in organic medium was ana-
lyzed as well. A scheme of the reactions assayed is showed in
Fig. 1.
3
7 °C, and p-NP in the supernatant was measured as
mentioned above. A reaction mixture without alcohol
served as hydrolysis control. In the absence of a biocata-
lyst, no reaction was observed. One unit of transesterifi-
cation activity was defined as the amount of biocatalyst that
released 1 lmol of p-NP per min. Specific transesterifica-
tion activity was expressed as milliunits per gram of dry
cell weight or protein for mycelium and lyophilized
supernatant, respectively.
2
.6 Statistical Analysis
3.1 Hydrolysis Reactions in Aqueous and Organic
Statistical analysis was performed using the Minitab (ver-
sion 14; Minitab Inc) software for Windows. Statistical
significance values of the means were evaluated using a
one-way analysis of variance. Subsequent comparisons
were performed using Tukey’s post hoc test. Results were
presented as the mean ± SD. Differences were accepted as
significant when P \ 0.05.
Media
The specific hydrolytic activity of three biocatalyst systems
such as the constitutive mycelium-bound lipase, the
induced mycelium-bound lipase and the lyophilized
induced lipase supernatant was determined against various
substrates in both aqueous and organic media. Interest-
ingly, substrate specificity changed depending on the
medium in which hydrolysis was carried out. In aqueous
medium all three biocatalysts showed highest specific
hydrolytic activities with middle-chain fatty acids esters
(p-NPCa and/or p-NPL); while, in organic medium the
biocatalysts preparations displayed highest specific hydro-
lytic activities with long-chain fatty acids esters (p-NPP
and/or p-NPS) (Table 1). However, the constitutive
mycelium-bound lipase exhibited the highest specific
3
Results and Discussion
Previously, it was reported an induced extracellular lipo-
lytic extract from A. niger MYA 135 that is very stable in
the presence of 50 % water-miscible organic solvents. This
lipase activity also retains around of 60 and 80 % of its
specific hydrolytic activity in aqueous medium after incu-
bation for 1 h at 37 °C with n-butanol and n-hexanol,
respectively [23]. In addition, both the induced and the
constitutive mycelium bound lipases are very stable in
reaction mixture containing methanol and ethanol. In fact,
the constitutive mycelium bound-lipase maintaining almost
hydrolytic activity (53.8 ± 2.7 mU/g ) in organic med-
dw
ium in the presence of p-NPS and the induced mycelium-
bound lipase exhibited the highest specific hydrolytic
activity (37.8 ± 3.8 mU/g ) in aqueous medium in the
dw
presence of p-NPL. Concerning the lyophilized induced
lipase supernatant, the highest specific hydrolytic activity
(5295.0 ± 0.5 mU/gprotein) was observed in organic med-
ium using p-NPP as substrate. Thus, differences in sub-
strate specificity may result from the fact that the catalysis
is heterogeneous, except for the case of the hydrolysis in
aqueous medium catalyzed by the lyophilized induced
lipase supernatant. Therefore, diffusional limitations may
change the specificity of lipases toward different substrates.
Various factors are involved in chain-length selectivity
of lipases. Substrate thermodynamic properties determine
the chemical reactivity and reaction equilibrium. These
properties notably depend on substrate chain-length, tem-
perature, solvent and the composition of the reaction
medium [7, 27]. Enzyme structure and molecular dynamics
are determinants for substrate specificity. Influence of the
lipase structure on the chain-length specificity has been
1
00 % of its specific hydrolytic activity in aqueous med-
ium after exposure by 1 h at 37 °C in ethanol [22]. This is
an important result because hydrophilic solvents can often
destabilize the lipase activity.
On the other hand, under the conditions employed for
hydrolysis, the only possible acceptor was water whereas
during transesterification both acceptors, alcohols and
water, competed in the nucleophilic attack of the acyl-
enzyme [24]. Moreover, alcohol, like water, is not only a
substrate but also a solvent that may influence thermody-
namic parameters of the reaction and enzyme activity. In
initial experiments, the performance of both the constitu-
tive and the induced mycelium-bound lipases were evalu-
ated in transesterifications of p-nitrophenyl palmitate with
different alcohols in the presence of n-hexane as a solvent
[
22, 25]. Additionally, an entrapped extracellular purified
123

1
364
C. M. Romero et al.
Reaction
Hydrolysis
S
1
P
1
S
2
P
2
Reaction medium
Aqueous
Ester (p-NP derivate)
Ester (p-NP derivate)
Ester (p-NP derivate)
(p-NP)
(p-NP)
H
H
2
2
O
O
FA
FA
Hydrolysis
Organic
Transesterification
(p-NP) Alcohol
Ester
Organic
Biocatalyst
1
2
3
-Constitutive mycelium-bound lipase
-Induced mycelium-bound lipase
-Lyophilized induced lipase supernatant
S
1
Acyl-donor: p-NPderivate
Acyl acceptor: H
P
1
2
O
Acyl acceptor: Alcohol
Product: p-NP
S
2
S
2
Product: FA
P
2
P
2
Product: Ester
Fig. 1 Mechanisms of hydrolysis and transesterification reactions catalyzed by lipase preparations
Table 1 Specific hydrolytic activities in both aqueous and organic media and RO/A ratio of all three biocatalysts from A. niger MYA 135
*
Biocatalysts/substrate
Specific hydrolytic activity
Aqueous medium (mU/gdw
R
O/A
)
Organic medium (mU/gdw)
Constitutive mycelium-bound lipase
p-NPCa (C10)
33.8 ± 0.5 (e)
31.9 ± 4.6 (e)
13.1 ± 1.1 (b)
7.5 ± 0.6 (a)
25.2 ± 0.3 (d)
16.4 ± 0.9 (c)
16.5 ± 2.1 (c)
53.8 ± 2.7 (f)
0.75
0.52
1.26
7.13
p-NPL (C12)
p-NPP (C16)
p-NPS (C18)
Induced mycelium-bound lipase
p-NPCa (C10)
20.0 ± 0.8 (c)
37.8 ± 3.8 (e)
19.5 ± 1.0 (c)
12.3 ± 0.2 (a)
15.1 ± 0.4 (b)
16.0 ± 1.0 (b)
25.9 ± 0.8 (d)
21.0 ± 0.2 (c)
0.76
0.42
1.32
1.71
*
p-NPL (C12)
p-NPP (C16)
p-NPS (C18)
Biocatalysts/substrate
Specific hydrolytic activity
R
O/A
Aqueous medium (mU/gprotein
)
Organic medium (mU/gprotein)
Lyophilized induced lipase supernatant
p-NPCa (C10)
3122.1 ± 72.8 (e)
4381.9 ± 31.5 (g)
2083.4 ± 35.9 (b)
1174.6 ± 97.1(a)
2798.8 ± 0.3 (c)
2843.1 ± 0.1 (d)
5295.0 ± 0.5 (h)
4089.9 ± 0.2 (f)
0.90
0.65
2.54
3.48
p-NPL (C12)
p-NPP (C16)
p-NPS (C18)
*
R
O/A, ratio of specific hydrolytic activity in organic medium to specific hydrolytic activity in aqueous medium. For each biocatalyst, values
with the same letter are not significantly different (P [ 0.05)
1
23

Specific Lipase-Catalyzed Hydrolysis
1365
1
0
0
.0
.8
.6
studied by several authors [28, 29]. The structural flexi-
bility of the enzyme is lower in organic solvents than in
aqueous media, which may also altered enzyme activity
[
4, 30]. This effect is especially important with lipases for
which the opening of the lid covering the active site is
necessary for good activity [31, 32]. Thus, in organic
medium a small amount of water molecules is frequently
essential to obtain a sufficient enzyme conformational
flexibility [33, 34].
0.4
0
0
.2
.0
0
.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
-
-
0.2
0.4
To simulate industrial application, the specific hydro-
lytic activity in n-hexane was determined without any
treatment or equilibration at a given water activity. In this
connection, the results obtained in this work can be useful
in the fatty acid industry where lipase-catalyzed hydrolysis
of fats and oils at mild temperature in organic solvent could
reduce energy cost, facilitate recovery of the fatty acid
product, and yield a product of superior quality [35].
-0.6
log (Activity in water)
Fig. 2 Log scale plot of RO/A versus activity in water for all three
biocatalysts from A. niger MYA 135. Each line represents the linear
regression of all experimental points. Constitutive mycelium-bound
lipase (diamond), Induced mycelium-bound lipase (square) and
lyophilized induced supernatant (triangle)
3
.2 Comparison of Specific Hydrolytic Activities
in Aqueous and Organic Media: The Activity Ratio
RO/A
mycelium-bound lipase and lyophilized induced lipase
supernatant were 0.39, 0.23 and 0.46, respectively. How-
ever, a log/log plot of RO/A of the three biocatalysts versus
2
Considering that the same hydrolytic reaction was con-
ducted in both aqueous and organic media the activity ratio
RO/A, defined as the ratio of activity in organic medium to
that in aqueous medium, was used to characterize the
activity in organic medium [9]. As shown in Table 1, RO/A
values from 0.42 to 7.13 were obtained. Although RO/A
values for all three biocatalysts were less than one during
hydrolysis of middle-chain fatty acid esters and higher than
one during hydrolysis of long-chain fatty acid esters, the
constitutive mycelium-bound lipase showed the highest
RO/A value (7.13) in a reaction mixture containing p-NPS as
substrate. It means that the constitutive biocatalyst prepa-
ration was the most active in hydrolysis reaction conducted
in organic medium. In addition, this RO/A value was higher
than that reported for a lipase activity from A. niger A6
AHA gave a satisfactory linear relationship (R : 0.85 and a
2
slope of -1.4 for constitutive mycelium-bound lipase, R :
0.87 and a slope of -1.2 for induced mycelium-bound
2
lipase and R : 0.92 and a slope of -1.3 for lyophilized
induced lipase supernatant) (Fig. 2). As can be seen, the
RO/A values decreased with the AHA, which shows the
influence of the substrate specificity besides the possible
impact of diffusional limitations.
3.3 Transesterification Reactions
Transesterifications of different alcohols with various
p-nitrophenyl derivatives using all three biocatalysts
preparations were evaluated in n-hexane (Fig. 3). For
methanolysis and ethanolysis, the constitutive mycelium-
bound lipase displayed an interesting preference for C16
substrate (Fig. 3A). Such mono alkyl esters like methyl and
ethyl palmitate are component of biodiesel, an alternative
fuel for diesel engines [36]. In addition, transesterifications
catalyzed by the induced mycelium-bound lipase in the
presence of propanol or butanol and long-chain fatty acid
esters (p-NPP and p-NPS) showed a good performance as
well. This is an interesting result because propanol and
butanol were also used in the biodiesel production due to
the good pouring characteristics of their fatty esters at low
temperatures [37].
(
Amano) [9], which showed a RO/A value of 0.3.
On the other hand, in a useful work, Pencreac h and
0
Baratti [9] reported the activity ratio RO/A calculated for 26
commercial lipase preparations. They found that this
parameter widely varies within the tested biocatalysts. In
this connection, the results presented in this work shown
that for a given substrate and solvent the RO/A ratio also
depend on the way that the biocatalyst was prepared.
Additionally, in agreement with those authors, the activity
in aqueous medium cannot be predicted from the activity in
organic media. This fact was confirmed by studying the
relationship between activity in aqueous (AHA) and organic
media (AHO) for each biocatalyst. A log/log plot of AHO
versus AHA showed a cloudy pattern with no real correla-
On the other hand, in general, the induced mycelium-
bound lipase displayed high specific transesterification
activities in the presence of water-miscible alcohols and
middle-chain fatty acid esters (p-NPCa and p-NPL),
being the highest specific transesterification activity
2
tion between the two variables (data not shown); the R
values for constitutive mycelium-bound lipase, induced
123

1
366
C. M. Romero et al.
Fig. 3 Specific
90
b
A
transesterification activities of
all three biocatalysts from
A. niger MYA 135 determined
in reaction mixtures containing
p-nitrophenyl derivatives and
different alcohols. Reaction
mixture without alcohol served
as hydrolysis control. Error
bars represent the standard
deviation calculated from at
least three independent
Constitutive mycelium-bound lipase
8
7
6
0
0
0
b
b
b
b
c
a
50
c
c
a
a
a
a
a
a
a
a
4
3
0
0
a
b
a
b
b
a a
experiments. For each alcohol,
bars with the same letter are not
significantly different
20
b
a
a
1
0
0
a
(
P [ 0.05)
Methanol
Ethanol
Propanol
d
Butanol
Hexanol
Heptanol
Control
1
00
B
Induced mycelium-bound lipase
b
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
b
c
b
b b
b
b
b
b
b
b
a
a
a
ab
a
a
a a
a
c
b
a
a
a
a
Methanol
Ethanol
Propanol
Butanol
Hexanol
Heptanol
Control
6
5
4
3
2
1
0000
0000
0000
0000
0000
0000
0
C
c
c
Lyophilized induced lipase supernatant
c
c
b
b
a
a
c
b
a a
Methanol
Ethanol
Propanol
Butanol
Hexanol
Heptanol
Control
p-Nitrophenyl caprate
p-Nitrophenyl laurate
p-Nitrophenyl stearate
p-Nitrophenyl palmitate
(
91.4 ± 1.7 mU/g ) observed in a reaction mixture con-
dw
highlight the good performance of this biocatalyst prepa-
ration in transesterifications carried out in the presence of
water-immiscible alcohols and long-chain fatty acid esters
(p-NPP and p-NPS).
taining propanol and p-NPL (C12) (Fig. 3B). Those syn-
thesized esters have applications as flavors constituents in
food industry [38]. Besides, it is also interesting to
1
23

Specific Lipase-Catalyzed Hydrolysis
1367
Finally, both p-NPC (C10) and p-NPL (C12) were
preferentially methanolized by the lyophilized induced
lipase supernatant (Fig. 3C). Thus, under transesterification
conditions, this lipase activity was the most specific bio-
catalyst preparation.
combined influence of enzyme and substrate properties.
According to the results discussed in this work, the sub-
strate specificity also depends on the way that the enzyme
was prepared.
In addition, the relation between the transesterification
and the hydrolysis in organic medium was also evaluated in
more detail. Each transesterification value was subtracting
to that measured for its respective hydrolysis control. This
transesterification value was used to analyze the ratio of
transesterification to hydrolysis in organic medium (AT/
AHO) for each biocatalyst (Table 2). Thus, in general, the
constitutive mycelium-bound lipase showed selectivity
toward synthesis when either p-NPL (C12) or p-NPP (C16)
was used as acyl donors. On the contrary, specific hydro-
lytic activity was favored when either p-NPCa (C10) or
p-NPS (C18) was used as acyl donor, which agrees with the
highest specific hydrolytic activities observed in organic
medium containing these p-nitrophenyl derivatives
(Table 1). On the other hand, the induced mycelium-bound
lipase exhibited the highest A /A ratio in the presence of
3
.4 Transesterification Versus Hydrolysis in Organic
Medium
An important issue in this study was the selectivity-based
analysis of each lipase preparation toward transesterifica-
tion or hydrolysis in organic medium. As displayed in
Fig. 3, of the 72 transesterifications tested, 45 exhibited a
specific transesterification activity value higher than its
corresponding hydrolysis control (reaction mixture without
alcohol). However, the selectivity toward a given reaction
differed with each biocatalyst preparation. The mycelium-
bound lipase showed around sixty percent of selectivity
toward transesterification; of 24 transesterifications tes-
ted, 15 revealed a specific transesterification activity
value higher than its corresponding hydrolysis control.
Interestingly, the reactivity toward transesterification was
drastically increased in the presence of the induced
mycelium-bound lipase; only three reactions with a specific
activity value lower than its corresponding hydrolysis
control were observed. Concerning the reaction catalyzed
by the lyophilized induced lipase supernatant, this biocat-
alyst preparation exhibited a remarkable specificity toward
transesterification, especially in the presence of methanol;
of 24 transesterifications tested, 15 reactions displayed a
specific transesterification activity value lower than its
corresponding hydrolysis control, showing that in most
cases water seems to be a better acyl acceptor than the
alcohols tested. In this connection, Vaysse et al. [10]
reported the chain-length selectivity profile of seven lipa-
ses. They found that the chain-length specificity depends
on the enzyme and the reaction considered, due to the
T
HO
p-NPL (C12) and propanol. Only with methanol as acyl
acceptor and p-NPP as acyl donor the reaction seems to be
completely shifted to hydrolysis; in fact, the highest spe-
cific hydrolytic activity in organic medium was observed in
the presence of p-NPP (Table 1). Finally, the lyophilized
induced lipase supernatant showed a clear preference
toward transesterification in reaction mixtures containing
methanol as acyl acceptor and middle-chain fatty acids
esters as acyl donors, showing an A /A ratio of 16.2 for
T
HO
p-NPCa and 17.9 for p-NPL (Table 2).
4 Conclusions
Three lipase preparations such as the constitutive myce-
lium-bound lipase, the induced mycelium-bound lipase and
the lyophilized induced supernatant obtained from A. niger
Table 2 Selectivity of all three biocatalysts from A. niger MYA 135 toward transesterification or hydrolysis in organic medium
a
T
A /AHO
Biocatalysts Constitutive mycelium-bound lipase
Induced mycelium-bound lipase
Lyophilized induced lipase supernatant
Substrate
p-NPCa
p-NPL
p-NPP
p-NPS
p-NPCa p-NPL p-NPP p-NPS p-NPCa
p-NPL
p-NPP
p-NPS
Methanol
Ethanol
–
0.4
0.4
0.8
\0.1
–
0.1
1.5
1.3
3.7
2.7
1.6
1.6
3.1
1.0
1.5
2.3
–
–
\0.1
3.7
3.0
2.9
4.7
3.1
1.2
1.0
–
0.2
0.4
2.6
2.6
1.5
1.7
16.2
–
18.0
–
2.7
0.7
0.6
–
0.8
–
–
0.4
0.6
1.6
1.1
1.5
Propanol
Butanol
Hexanol
Heptanol
–
2.8
–
–
–
0.2
–
1.2
–
–
–
1.0
–
–
–
–
–
1.0
6.7
6.9
1.7
0.4
Each transesterification value was subtracting to that measured for its respective hydrolysis control. This transesterification value was used to
analyze the ratio A
T
/AHO
a
T
A /AHO, ratio of specific transesterification activity to specific hydrolytic activity in organic medium
123

1
368
C. M. Romero et al.
MYA 135 were evaluated based on their reactivity under
both hydrolytic and synthetic conditions. The specific
hydrolytic activity of all three biocatalyst was determined
against several p-nitrophenyl derivatives in both aqueous
and organic media. Interestingly, substrate specificity
changed depending on the medium in which hydrolysis was
carried out. Concerning the specific transesterification
activity, the induced mycelium-bound lipase was the bio-
catalyst most reactive, whereas the lyophilized induced
supernatant was the biocatalyst most specific. Finally, an
important issue in this study was the selectivity-based
analysis of each lipase preparation toward transesterifica-
tion or hydrolysis in organic medium. It was found that the
substrate specificity also depends on the way that the
enzyme was prepared. Thus, knowledge about substrate
specificity of a lipase preparation would simplify the
choice of a specific biocatalyst for a certain reaction and
optimize the industrial process.
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