Evaluation of styrene-divinylbenzene beads as a support to immobilize lipases

Article abstract of DOI:10.3390/molecules19067629

A commercial and very hydrophobic styrene-divinylbenzene matrix, MCI GEL CHP20P, has been compared to octyl-Sepharose beads as support to immobilize three different enzymes: lipases from Thermomyces lanuginosus (TLL) and from Rhizomucor miehie (RML) and Lecitase Ultra, a commercial artificial phospholipase. The immobilization mechanism on both supports was similar: interfacial activation of the enzymes versus the hydrophobic surface of the supports. Immobilization rate and loading capacity is much higher using MCI GEL CHP20P compared to octyl-Sepharose (87.2 mg protein/g of support using TLL, 310 mg/g using RML and 180 mg/g using Lecitase Ultra). The thermal stability of all new preparations is much lower than that of the standard octyl-Sepharose immobilized preparations, while the opposite occurs when the inactivations were performed in the presence of organic co-solvents. Regarding the hydrolytic activities, the results were strongly dependent on the substrate and pH of measurement. Octyl-Sepharose immobilized enzymes were more active versus p-NPB than the enzymes immobilized on MCI GEL CHP20P, while RML became 700-fold less active versus methyl phenylacetate. Thus, the immobilization of a lipase on this matrix needs to be empirically evaluated, since it may present very positive effects in some cases while in other cases it may have very negative ones.

Full text of DOI:10.3390/molecules19067629

Molecules 2014, 19, 7629-7645; doi:10.3390/molecules19067629
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Evaluation of Styrene-Divinylbenzene Beads as a Support to
Immobilize Lipases
1
2
3
1,4
Cristina Garcia-Galan , Oveimar Barbosa , Karel Hernandez , Jose C. S. dos Santos ,
5
1,
Rafael C. Rodrigues and Roberto Fernandez-Lafuente *
1
Departamento de Biocatalisis, ICP-CSIC, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain;
E-Mails: c.garcia@icp.csic.es (C.G.-G.); jscleiton@gmail.com (J.C.S.D.S.)
2
Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM),
Edificio Camilo Torres 210, Universidad Industrial de Santander, Bucaramanga 680001, Colombia;
E-Mail: oveimar@gmail.com
3
Biotransformation and Bioactive Molecules Group, Instituto de Química Avanzada de Cataluña-
CSIC Jordi Girona 18-26, 08034 Barcelona, Spain; E-Mail: khs27883@gmail.com
4
Departamento de Engenharia Química, Universidade Federal do Ceará, Campus do Pici, CEP
6
0455-760 Fortaleza, CE, Brazil
5
Biotechnology, Bioprocess and Biocatalysis Group, Institute of Food Science and Technology,
Federal University of Rio Grande do Sul, Av. Bento Gonçalves, 9500, P.O. Box 15090,
ZC 91501-970, Porto Alegre, RS, Brazil; E-Mail: rafaelcrodrigues@ufrgs.br
*
Author to whom correspondence should be addressed; E-Mail: rfl@icp.csic.es;
Tel.: +34-915-854-809; Fax: +34-91-854-760.
Received: 3 April 2014; in revised form: 3 June 2014 / Accepted: 4 June 2014 /
Published: 10 June 2014
®
Abstract: A commercial and very hydrophobic styrene-divinylbenzene matrix, MCI GEL
®
CHP20P, has been compared to octyl-Sepharose beads as support to immobilize three
different enzymes: lipases from Thermomyces lanuginosus (TLL) and from Rhizomucor
®
miehie (RML) and Lecitase Ultra, a commercial artificial phospholipase. The
immobilization mechanism on both supports was similar: interfacial activation of the
enzymes versus the hydrophobic surface of the supports. Immobilization rate and loading
®
®
capacity is much higher using MCI GEL CHP20P compared to octyl-Sepharose (87.2 mg
®
protein/g of support using TLL, 310 mg/g using RML and 180 mg/g using Lecitase
Ultra). The thermal stability of all new preparations is much lower than that of the standard
®
octyl-Sepharose immobilized preparations, while the opposite occurs when the
inactivations were performed in the presence of organic co-solvents. Regarding the

Molecules 2014, 19
7630
hydrolytic activities, the results were strongly dependent on the substrate and pH of
®
measurement. Octyl-Sepharose immobilized enzymes were more active versus p-NPB
®
than the enzymes immobilized on MCI GEL CHP20P, while RML became 700-fold less
active versus methyl phenylacetate. Thus, the immobilization of a lipase on this matrix
needs to be empirically evaluated, since it may present very positive effects in some cases
while in other cases it may have very negative ones.
Keywords: lipase immobilization, modulation of lipase activity, interfacial activation,
styrene divinylbencene matrix
1
. Introduction
Lipases are the most used enzymes in biocatalysis, both at the industrial and academic levels [1–5].
This is because lipases are quite robust under a wide range of conditions and reaction media (including
organic solvents, ionic liquids or supercritical fluids) [6,7], have a broad specificity accepting very
different substrates (while presenting in some instances a high regio and enantio selectivity and
specificity) [8,9], and can catalyze many interesting reactions (hydrolysis of esters, esterification,
transesterification) [10–13] and even some promiscuous reactions (perhydrolysis, carbon-carbon bonds
formation) [14–16].
Even with these good initial prospects, the properties of lipases need to be improved, as for many
other enzymes, to be used as industrial biocatalysts. Enzyme immobilization, if properly utilized, may
improve many features, from stability to activity and specificity [17–22]. In some cases, the one-step
immobilization-purification of the enzyme may increase the impact of the immobilization step,
because in industry, pure enzymes are hardly utilized and in some cases, this may generate some
problems [23].
Lipases present a peculiar catalytic mechanism, called interfacial activation [24–26]. In most cases,
their active center is isolated from the medium by a polypeptide chain called lid (closed form) [27].
In some cases, the lid is so small that it cannot seclude the active center from the medium, as it is the
case of the lipase B from Candida antarctica (CALB) [28]. In most cases, the lid is able to fully isolate
the active center, and even in some cases a double lid has been described [29]. The internal face of the
lid is hydrophobic and is interacting with the hydrophobic areas around the active center. The lid can
move, exposing the active center as well as this large hydrophobic pocket, in an equilibrium shifted
towards the closed form [24–26]. In the presence of drops of the natural substrate (oils), the
hydrophobic pocket of the open form is stabilized after enzyme adsorption and the enzyme becomes
adsorbed and oriented on the substrate. It has been shown that lipases are adsorbed on any hydrophobic
surface, including hydrophobic proteins [30], open forms of other lipase molecules [31,32], or
hydrophobic supports [33].
The immobilization of lipases on hydrophobic supports has been reported as a very efficient way to
immobilize, purify and hyperactivate lipases, allowing retention of the open form of the enzyme
without any external interface [33]. This way, the lipases immobilized following this strategy will have
the open structure stabilized by interactions with the support, involving the hydrophobic area of the lid

Molecules 2014, 19
7631
and the hydrophobic areas surrounding the active center [33]. It has also been shown that the nature
internal morphology, hydrophobicity of the surface, etc.) of the support may greatly affect the final
(
properties of the immobilized enzyme (activity, stability, even selectivity and specificity) [34,35].
®
Recently, a commercial matrix composed of styrene divinylbenzene (MCI GEL CHP20P) allowed
great improvement of some properties of the lipase B from Candida antarctica (CALB) [36], perhaps
the most popular enzyme in biocatalysis [37]. Enzyme activity versus many substrates could be
improved when comparing this immobilized catalyst to the commercial one or even other home-made
preparation based on similar immobilization mechanism. Stability in co-solvents was improved, while
thermo-stability was lowered using this very hydrophobic matrix [36]. The loading capacity of the
support using CALB was also very high. These immobilized biocatalysts were used in other reactions
exhibiting in many cases some advantages: in some instances higher activity was found, in other
instances a better operational stability was detected [38–41]. Even after this preliminary success using
CALB, few examples on the use of this support to immobilize other enzyme may be found in the
literature. The lipase from Thermomyces lanuginosus has been utilized in esterification reactions after
immobilization on this support [42], but the biocatalyst properties (activity, stability, loading capacity),
have not been analyzed. In another paper, the purification/immobilization of the lipase from
Staphylococcus warneri EX17 on this support was shown [43].
Now, in this new paper, we intended to present the prospects of this new support to immobilize the
lipases from Thermomyces lanuginosus (TLL) [44] and the lipase from Rhizomucor miehei (RML) [45,46],
®
very likely some of the most popular lipases after CALB. We have also included Lecitase Ultra (LU)
in these studies, a commercial chimeric phospholipase built from the gen of the lipase from
Thermomyces lanuginosus (to obtain good stability) and that of the phospholipase from Fusarium
®
oxysporum (to get the phospholipase activity) [47]. As reference, we will use octyl-Sepharose beads,
a support reported as very useful to immobilize lipases via this strategy [48]. The loading capacity of
the support and activity versus several substrates, as well as the stability in diverse conditions was
analyzed for all biocatalysts to advance on the prospects of this matrix as a general support to
immobilize lipases.
2
. Results and Discussion
2
.1. Immobilization Courses of the Different Lipases on Both Supports
®
Figure 1 shows the immobilization courses of the three enzymes on octyl-Sepharose and MCI
®
®
GEL CHP20P beads. The immobilization was always faster using MCI GEL CHP20P, in some
cases differences are clearer (TLL), in other cases the differences are lower (RML). In fact, using TLL
the immobilization yield using octyl support is around 80% after 24 h, while using the new support the
yield is 100% just after some few minutes of contact. This should be related to the more hydrophobic
nature of this support, the support must make a competition with the other lipase molecules, that can
also form bimolecular aggregates via interaction with two open forms of the lipase [31,49] to
immobilize the enzymes. In Table 1 the values of the loading capacity of the new support are given,
considering a maximum immobilization time of 24 h. The loading using the new supports (87.2 mg

Molecules 2014, 19
7632
®
protein/g of support using TLL, 310 mg/g using RML and 180 mg/g using Lecitase Ultra) is nearly
®
thirty-fold higher than that observed using octyl-Sepharose .
®
Figure 1. Immobilization courses of different enzymes on octyl-Sepharose and MCI
®
GEL CHP20P beads. The activity of the supernatants is shown (free enzyme remained
fully active under immobilization conditions). Experiments were carried out as described in
methods, using a support/total volume ratio of 0.1 and a loading of 3 mg of protein/g of
®
®
support. A: TLL, B: RML, C: LU. Circles: octyl-Sepharose , Squares: MCI GEL
CHP20P. The results are the mean of 3 independent experiments and the experimental
error was never over 10%.
100
1
00
8
0
0
8
6
4
2
0
0
0
0
0
6
40
20
0
0
10
20
30
0
10
20
30
Time, (h)
Time, (h)
(
a)
(b)
100
80
60
40
20
0
0
10
20
30
Time, (h)
(
c)
®
®
Table 1. Loading observed using the different enzymes on octyl-Sepharose and MCI GEL CHP20P.
®
®
Lipase/support
Octyl-Sepharose
11 ± 2
MCI GEL CHP20P
RML
TLL
LU
310 ± 20
90 ± 8
20 ± 2
30 ± 3
180 ± 15
The loadings are given in mg of protein per gram of wet support. Experiments were performed as described
in Experimental section. The results are the mean of three independent experiments and the experimental
error was never over 10%.

Molecules 2014, 19
.2. Effect of pH on Immobilized Enzyme Activities Versus pNPB
7633
2
®
All studied lipases are hyperactivated after immobilization on octyl-Sepharose , RML multiplied
®
the activity by 3.8, TLL by 1.65 and Lecitase Ultra by 2 (results not shown). The increase in lipase
®
activity after immobilization on octyl-Sepharose has been previously reported and it has been
explained by the stabilization of the open form of the lipase [48]. The observed increment in activity
after the immobilization depends on the loading of the immobilized biocatalyst (because diffusion
problems may decrease the observed activity after immobilization) and the concentration of the free
lipase in the activity measurements (because this determines the dimerization of the lipase and can
cause an alteration of the detected activity ), etc. [31,49].
Table 2 shows the activity data for the 3 enzymes immobilized on both supports in a pH range from
5
to 10, using a loading of only 3 mg/g of support. It should be considered that the free enzymes have a
tendency to produce aggregates, therefore they may generate results, which could be very hard to
®
interpret [32,50]. RML activity is around 20-fold lower when immobilized on MCI GEL CHP20P
®
than when immobilized on octyl-Sepharose . However, the pH/activity curve is rather similar. TLL
®
activity is also lower using MCI GEL CHP20P, but the differences are shorter. The optimal pH value
is similar for both biocatalysts. Nevertheless, the new biocatalyst seems to retain more activity at
®
alkaline pH values. The results were even more negative using Lecitase , the drop in activity was of
®
®
more than 100 fold factor. Moreover, the optimal pH was 6 for MCI GEL CHP20P-Lecitase , and 10
for the octyl preparation.
Table 2. Specific activities of the different biocatalyst versus pNPB under different conditions.
pH
Biocatalyst
5
6
7
8
9
10
38
TLL-OS
TLL-MCI
RML-OS
RML-MCI
LU-OS
26
33
34
35
55
1.38
9.7
1.39
11.25
0.45
13
1.41
12.1
0.50
11.5
0.42
3.72
10.2
0.47
16.5
0.37
4.66
6.4
4.44
1.3
0.45
10.5
0.16
0.33
16.8
0.36
0.02
28
LU-MCI
0.50
0.45
For a better comparison, loadings of 3 mg of protein/g of wet support were used in all cases. OS: Octyl
®
®
Sepharose ; MCI: MCI GEL CHP20P. Specific activity is defined as μmol of pNP released per minute and
mg of immobilized enzyme. Experiments were performed as described in methods section. The results are the
mean of three independent experiments and the experimental error was never over 7%.
It is not easy to explain this difference in activity versus pNPB using the new support and
®
octyl-Sepharose , considering that in both cases a similar immobilization mechanism is expected,
involving interfacial activation versus the hydrophobic support [48]. It is also curious that the
pH/activity profile may have some clear differences. The main difference between both supports is the
®
internal morphology (trunks for agarose, tunnels for MCI GEL CHP20P). In the MCI support,
adsorption takes place directly on the matrix and not in a hydrophobic layer over a hydrophilic matrix,
®
which makes MCI much more hydrophobic than octyl-Sepharose . Moreover, in the MCI support the
enzyme is interacting with the surface, not with acyl groups that can place some distance between

Molecules 2014, 19
7634
enzyme and support wall. Thus, one possible explanation is the occurrence of some partition effect of
the substrate, which is an aqueous solution, from the hydrophobic matrix of MCI, leading to lower
activity on this support. Additionally, it has been previously reported that the differences on the
immobilization support may be enough to explain differences on lipases immobilized via interfacial
activation on hydrophobic supports [35]. This way, it is expected that some conformational changes
induced by the very hydrophobic surface will lead to different activities [20,51].
®
®
2
.3. Thermal Stability of MCI GEL CHP20P and octyl-Sepharose Lipase Preparations
®
Table 3 shows the negative effect of the immobilization on MCI GEL CHP20P of all the enzymes
on their thermal stability. In general, differences in half-lives were shorter at pH 9 and much higher at
®
pH 5 and 7. The MCI preparations fully lost the activity under conditions where the octyl-Sepharose
preparations retained a high percentage of the initial activity; this was more dramatic using TLL
®
(
the difference in stability is so large that it cannot be compared), while using RML and Lecitase
Ultra differences, even very significant, are not so high (e.g., the stability of the new preparation for
®
Lecitase is more than fifteen folds lower at pH 5, twenty at pH 7 and less than two fold at pH 9).
®
This negative effect of the MCI GEL CHP20P on the thermal stability of the enzymes was also
found using CALB [36], and was explained due to their very hydrophobic nature, that may facilitate
the stabilization of partially unfolded enzyme structures [23,52].
Table 3. Thermal stability of the different biocatalysts at different pH values and 45 °C
(
given as half-lives in min).
pH
7
Biocatalyst
5
9
TLL-OS *
TLL- MCI
RML-OS
>90% after 10h >90% after 10h >90% after 10h
23
1800
180
510
30
20
750
105
900
45
23
26
30
30
24
RML-MCI
LU-OS **
LU-MCI **
®
®
Experiments were performed as described in the methods section. OS: Octyl-Sepharose ; MCI: MCI GEL
CHP20P. * At 45 °C the inactivation rate was too slow to calculate half live for the preparation TLL-OS; **
Temperature was increased to 50 °C to accelerate the inactivation. The results are the mean of three
independent experiments and the experimental error was never over 10%.
®
®
2
.4. Stability of MCI GEL CHP20P and octyl-Sepharose Lipase Preparations in Presence of
acetonitrile
Figure 2 shows the effect of the incubation in the presence of acetonitrile on the activity of the
different immobilized enzymes (measured in aqueous medium).

Molecules 2014, 19
Figure 2. Effect of the incubation in the presence of acetonitrile of different lipase
7635
preparations on the enzyme activity. Experiments were carried out as described in methods
at 25 °C and pH 7. (a): TLL (80% acetonitrile); (b): RML (30% acetonitrile); (c):
®
®
®
Lecitase . (30% acetonitrile) Circles: octyl-Sepharose , Squares: MCI GEL CHP20P.
The results are the mean of 3 independent experiments and the experimental error was
never over 5%.
1
00
1500
1250
1000
7
5
750
500
250
0
50
2
5
0
0
30
60
90
120
150
180
0
6
12
18
24
Time, (h)
Time, (h)
(
a)
(b)
3
2
2
1
1
00
50
00
50
00
50
0
0
4
8
12
16
20
24
Time, (h)
(
c)
Using TLL, both preparations were very stable on high concentrations of acetonitrile. In fact, the
incubation in 80% acetonitrile produced a very significant and progressive initial increase in activity
®
when the enzyme was immobilized on MCI GEL CHP20P, even by a 12-fold factor after 1–2 h.
®
Using octyl-Sepharose , there was also an increase in activity, but it is much lower: under a 40% after
2
h. After 1 week of incubation at this very high cosolvent concentration, the new preparation
®
presented almost 8 times more activity than the initial one, while the octyl-Sepharose TLL activity
had decreased to 90%.
Using RML, the incubation in 30% acetonitrile was enough to cause a decrease in enzyme activity
for both immobilized enzymes, with a slower decrease in enzyme activity using the new preparation
®
®
(
half-lives of 0.5 h for octyl-Sepharose -RML and 10 h for MCI GEL CHP20P-RML) (Figure 2).
®
Using Lecitase , the drop in activity was very rapid and similar for both preparations in 50%
®
®
acetonitrile, while using 30% of solvent MCI GEL CHP20P-Lecitase Ultra exhibited an initial
increase in activity followed by a progressive decrease in activity (Figure 2). This initial
®
®
hyperactivation was not observed using octyl-Sepharose . After 6 h, while the octyl-Sepharose
preparation maintained only 50% of the initial activity, the new preparation exhibited more than 100%.

Molecules 2014, 19
7636
This higher stability in the presence of organic solvent of the lipases immobilized on the styrene
divinylbenzene matrix can be explained by the higher strength of the enzyme support-interaction,
which cannot be broken by the concentration of the used organic solvents. The initial increase in
activity for some of the lipases suggests that the adsorption to the support may be too strong, favoring
a partial blocking of the active center, and that the presence of the solvents can produce some relaxing
on this adsorption, producing the reported initial increase of activity. However, direct conformational
changes of the enzyme structure cannot be discarded [20].
2
.5. Enzyme Activity Versus other Substrates
As it has been found in many different examples, the immobilization may greatly alter the enzyme
specificity, mainly using lipases, even if the mechanism of immobilization is similar [17,19,20]. For
this reason, the activities of the enzyme derivatives versus three structurally different substrates were
evaluated. First, a hydrophobic aliphatic ester, ethyl hexanoate was used. Second, methyl
phenylacetate, where the acid is an aromatic cycle, was assayed. And finally, the most complex one,
racemic methyl mandelate, that presents an aromatic ring and a hydroxyl group in alpha position
regarding the carboxyl group, was utilized as substrate. For TLL and RML; the commercial
preparations were used to compare the final activities, even though the home-made preparation
presented only 50 mg/g of support of enzyme to reduce the diffusion problems (around 20%–30% of
the maximum loading, as shown in Table 1).
Table 4 shows the specific activities of the different lipase preparations under different conditions.
Analyzing the results using TLL, the specific activity of the new preparation on the aliphatic ester
®
remained lower than that observed using octyl-Sepharose -TLL at pH 5 (almost 4-fold) or pH 7
(about 7-fold). However, at pH 8.5 the new preparation has a slightly higher specific activity. Using
the ester of phenylacetic, the new preparation is around 3 times less active than the octyl-Sepharose®
per mg of enzyme. However, using mandelic acid, the preparation becomes the most active, 2.5 fold at
pH 5 and 7, and 5.5 at pH 8.5. Even the optimal pH value depends on the substrate and the biocatalyst,
and that occurred even though none of the used compounds has ionizable groups in the range of the
utilized conditions. The highest activity was found at pH 5 using the aliphatic ester for both
biocatalysts, at pH 7 using all other combinations, except methyl mandelate and the new biocatalyst
that presented the highest activity at pH 8.5.
®
Comparing with the commercial preparation (Table 5), Lipozyme TL-IM, and using ethyl
hexanoate as substrate, the mass activity of the new biocatalyst is around 5-6 folds higher than that of
the commercial one, depending on the pH value. Using methyl phenyl acetate the differences are very
short, while using methyl mandelate, the differences are large: 90-folds at pH 5 and 7 and over 250 at
pH 8.5, favoring the new catalyst.
®
In the case of RML, the immobilization on MCI GEL CHP20P-RML produced a decrease of the
activity versus ethyl hexanoate at pH 7 (35-folds), and a little decrease at pH 7 (2-folds) while at pH
8
.5 is 50% more active (Table 4). Using methyl phenylacetate the new preparation is 7-fold more
®
active at pH 7, 3.5-fold at pH 5 and the octyl-Sepahrose -RML has no detectable activity at pH 8.5,
while using the new biocatalyst the activity was similar to that found at pH 7.

Molecules 2014, 19
Table 4. Specific activity of the different biocatalysts versus ethyl hexanoate (EH), methyl
7637
phenylacetate (MP) and methyl mandelate (MM) under different conditions.
pH
Biocatalyst/substrate
5
480
125
0.12
0.03
4.8
7
165
25
8.5
17
TLL-OS/EH
TLL- MCI/EH
TLL-OS/MP
TLL- MCI/MP
TLL-OS/MM
TLL- MCI/MM
RML-OS/EH
RML-MCI/EH
RML-OS/MP
RML-MCI/MP
RML-OS/MM
RML-MCI/MM
LU-OS/EH
20
0.21
0.06
6
0.09
0.03
3.1
10
16
17.5
0.13
0.2
671
333
0.02
0.07
4.5
435
13
−
3
0.03
2.1
<10
2.24
4.5
6.2
15
105
0.66
30
142
0.1
0.04
32
LU-MCI/EH
LU-OS/MP
29
<0.01
4.41
0.55
5.8
<0.01
4.5
<0.001
10
LU-MCI/MP
LU-OS/MM
1.35
55
11
LU-MCI/MM
47
Experiments were performed as described in the Experimental section. Activity is given in nmol of ester
®
®
hydrolyzed per minute and per mg of enzyme. OS: Octyl-Sepharose ; MCI: MCI GEL CHP20P.
®
Table 5. Activity per gram of wet biocatalyst of the MCI GEL CHP20P-lipase
preparations (50 mg/g of support) compared to the activity of the respective commercial
preparations.
pH
Biocatalyst/Substrate
5
1133
6325
15
7
8.5
150
1010
9
IM-TLL/EH
TLL- MCI/EH
IM-TLL/MP
186
1249
29
TLL- MCI/MP
IM-TLL /MM
TLL- MCI/MM
IM-RML/EH
RML-MCI/EH
IM-RML/MP
RML-MCI/MP
IM-RML/MM
RML-MCI/MM
14
31.5
8.5
13.5
3.2
6.25
510
985
16650
7820
3.7
800
78
870
17
642
2310
105
490
5250
10.2
7900
112
590
7100
75
712
Experiments were performed as described in the Exprimental section. Activity is given in nmol of ester
®
®
hydrolyzed per minute and per wet gram of support. IM-TLL (Lipozyme TL-IM) and IM-RML (Lipozyme
RM-IM) were products from Novozymes, MCI preparations are those obtained by immobilization on MCI
®
GEL CHP20P. EH: ethyl hexanoate; MP: methyl phenylacetate; MM: methyl mandelate.

Molecules 2014, 19
7638
Using the most complex substrate, methyl mandelate, the activity kept a similar trend but the
differences are higher in favor of the new preparation; at pH 7 the new preparation is 17 fold more
active, at pH 8.5 is over 30 times more active, while at pH 5 differences become reduced (3-fold).
®
Compared to the commercial preparation (Table 5), Lipozyme RM-IM, the differences are huge
and dependent on the substrate and the pH. Using the aliphatic ester, the new preparation is around
7–8 folds more active at pH 5 and 7, while at pH 8.5 have a 70% more activity. Using methyl
phenylacetate, the commercial preparations is much more active than the new preparation: at pH 7 it is
more than 20 folds, but at pH 5 it is over 2000 folds and at pH 8.5 more than 70 times. This situation is
®
reversed using methyl mandelate, the MCI GEL CHP20P-RML is over 8–12 fold more active,
depending on the pH value.
®
®
In the case of Lecitase Ultra (Table 4), the immobilization of the enzyme on MCI GEL CHP20P,
the three substrates permitted a much higher activity than the enzyme immobilization on octyl-Sepharose®,
in opposition to the results using pNPB. Using ethyl hexanoate, the activity increased by a factor of 45
(
at pH 7), 800 (at pH 5) or 3000 (at pH 8.5). Using methyl phenylacetate, the activity could not be
®
®
®
®
determined using octyl-Sepharose Lecitase , while the activity using MCI GEL CHP20P-Lecitase
was around 10-fold lower than using the aliphatic ester (at leats difference in activity was a 5000 fold
factor). Using methyl mandelate, differences in activity were not so large, at pH 7, the new preparation
was 40 fold more active, while at pH 5, it was more than 10 folds more active, and at pH 8.5 it was
only 4.5 fold more active.
3. Experimental
3.1. Materials
The enzymes used in this work were Thermomyces lanuginosus lipase free or immobilized in a
®
silicate support (Lipozyme TL-IM); Rhizomucor miehei lipase free or immobilized in an
®
®
anion-exchange resin (Lipozyme RM-IM) and free Lecitase Ultra. All of them were kindly
®
donated by Novozymes (Madrid, Spain). Octyl-Sepharose crosslinked 4% beads were from GE
®
Healthcare (Uppsala, Sweden). Styrene–divinylbenzene MCI GEL CHP20P beads p-nitrophenyl
butyrate (p-NPB), methyl mandelate, ethyl hexanoate, and methyl phenylacetate were from Sigma
Chemical Co. (St. Louis, MO, USA). All experiments have been performed, at least, by triplicate and
the values are the mean values. Standard error was under 10% in all cases.
3.2. Standard Determination of Enzyme Activity
This assay was performed by measuring the increase in absorbance at 348 nm (isosbestic point)
produced by the released p-nitrophenol in the hydrolysis of 0.4 mM p-nitrophenyl butyrate (p-NPB)
−1
−1
in 100 mM sodium phosphate at pH 7.0 and 25 °C (ε under these conditions is 5,150 mol ·cm ).
To start the reaction, 50–100 µL of lipase solution or suspension was added to 2.5 mL of substrate
solution. One unit of activity (U) was defined as the amount of enzyme that hydrolyzes 1 µmol of
p-NPB per minute under the conditions described previously. Protein concentration was determined
using Bradford’s method [53] and bovine serum albumin was used as the reference.

Molecules 2014, 19
7639
In the studies of pH effects on the enzyme activity, the protocol was similar but the buffer in the
measurements was changed according to the pH value: sodium acetate at pH 5, sodium phosphate at
pH 6–8 and sodium borate at pH 9–10. At 25 °C, all the preparations remained fully active after
incubation for several hours at any of these pH values.
3
.3. Immobilization of Lipases
Styrene-divinylbenzene support is so hydrophobic that water hardly can directly penetrate into their
pores. Thus, a sample of 10 g of this support was suspended in 100 mL of acetonitrile for 1 h under
mild stirring, and then 100 mL of water were added. After 1 additional hour of mild stirring, the
supports were filtered and re-suspended in 100 mL pure water for another hour under stirring. Finally,
the supports were washed in a glass funnel five times with 5 volumes of water and stored at 4 °C.
Immobilization was performed using 3 or 50 mg of protein per g of wet support. The commercial
samples of the lipases were diluted in the corresponding volume of 5 mM sodium phosphate at pH 7.
Then, the supports were added. The activity of both supernatant and suspension was followed using
p-NPB (see Section 3.2). After immobilization the suspension was filtered and the supported lipase
was washed several times with distilled water.
3
.4. Determination of the Loading Capacity of the Different Supports
The different supports (2 g) were added to a 400 mL solution of the corresponding lipase solution
having from 0.1 to 1 mg protein/mL in 5 mM sodium phosphate at pH 7 and 25 °C, adding 0.01%
sodium azide to prevent microbial contamination. Samples of supernatant were taken periodically to
measure its activity by the p-NPB assay and immobilization was considered to be complete when no
significant changes in enzyme activity in the supernatant were detected after 4 h, with a maximum
of 24 h.
3
.5. Thermal Inactivation of Different Lipase Immobilized Preparations
To check the stability of enzyme derivatives, immobilized enzyme (1 g) was suspended in 10 mM
of sodium acetate (5 mL) at pH 5, sodium phosphate at pH 7 or sodium carbonate at pH 9 at the
indicated temperatures. Periodically, samples were withdrawn and the activity was measured using
p-NPB. Half-lives were calculated from the observed inactivation courses.
3
.6. Inactivation of Different Enzyme Preparations in the Presence of Acetonitrile
Enzyme preparations were incubated in mixtures of acetonitrile in 100 mM Tris–HCl at pH 7
and 25 °C (pH previously adjusted using NaOH) to proceed with the inactivation. Periodically,
samples were withdrawn and the activity was measured using p-NPB. The acetonitrile present in the
samples had no significant effect during enzyme activity determination.

Molecules 2014, 19
.7. Hydrolysis of Ethyl Hexanoate
7640
3
Enzyme activity was determined by using ethyl hexanoate; 200 mg of the immobilized preparations
were added to 0.6 mL of 25 mM substrate in 50 mM buffer containing 50% CH CN. The buffer was
3
sodium acetate at pH 5, sodium phosphate at pH 7 and sodium carbonate at pH 8.5. All experiments
were carried out at 25 °C under continuous stirring. The conversion degree was analyzed by RP-HPLC
(Spectra Physics SP 100 coupled with a Spectra Physics SP 8450 UV detector, (Thermo, Santa Clara,
CA, USA) using a Kromasil C18 (15 cm × 0.46 cm) column. Samples (20 μL) were injected and eluted
at a flow rate of 1.0 mL/min using acetonitrile /10 mM ammonium acetate aqueous solution (50:50,
v/v) and pH 3.2 as mobile phase and UV detection was performed at 208 nm. Hexanoic acid has a
retention volume of 3.4 mL while ester has a retention volume of 14.2 mL. One unit of enzyme activity
was defined as the amount of enzyme necessary to produce 1 nmol of hexanoic acid per minute under
the conditions described above. Activity was determined by triplicate with a maximum conversion of
2
0%–30%, and data are given as average values.
3
.8. Hydrolysis of Methyl Phenylacetate
Enzyme activity was determined by using methyl phenylacetate; 200 mg of the immobilized
preparations were added to 0.6 mL of 5 mM substrate in 50 mM buffer containing 50% CH CN.
3
The buffer was sodium acetate at pH 5, sodium phosphate at pH 7 and sodium carbonate at pH 8.5.
All experiments were carried out at 25 °C under continuous stirring. The conversion degree was
analyzed by RP-HPLC (Spectra Physics SP 100 coupled with a Spectra Physics SP 8450 UV detector)
using a Kromasil C18 (15 cm × 0.46 cm) column. Samples (20 μL) were injected and eluted at a flow
rate of 1.0 mL/min using acetonitrile, 10 mM ammonium acetate aqueous solution (35:65, v/v) and pH 2.8,
as mobile phase and UV detection was performed at 230 nm. Phenylacetic acid has a retention volume
of 4.2 mL while the ester has a retention volume of 12.5 mL. One unit of enzyme activity was defined
as the amount of enzyme necessary to produce 1 nmol of phenylacetic acid per minute under the
conditions described above. Activity was determined by triplicate with a maximum conversion
of 20%–30%, and data are given as average values.
3.9. Hydrolysis of Methyl Mandelate
Enzyme activity was also determined by using methyl mandelate. The immobilized preparations
(
200 mg) were added to 50 mM substrate in 50 mM sodium acetate (1 mL) at pH 5, 50 mM sodium
phosphate at pH 7 or 50 mM sodium carbonate at pH 8.5 and 25 °C under continuous stirring. The
conversion degree was analyzed by RP-HPLC (Spectra Physics SP 100 coupled with a Spectra Physics
SP 8450UV detector) using a Kromasil C18 (15 cm × 0.46 cm) column. Samples (20 μL) were injected
and eluted at a flow rate of 1.0 mL/min using acetonitrile/ 10 mM ammonium acetate (35:65, v/v) at
pH 2.8 as mobile phase and UV detection was performed at 230 nm. The acid has a retention volume
of 2.4 mL while the ester has a retention volume of 4.2 mL One unit of enzyme activity was defined as
the amount of enzyme necessary to produce 1 nmol of mandelic acid per minute under the conditions
described above. Activity was determined by triplicate with a maximum conversion of 20%–30%, and
data are given as average values.

Molecules 2014, 19
7641
4
. Conclusions
®
®
MCI GEL CHP20P beads are a support that permits very high lipase (or Lecitase Ultra) loading
and a very rapid immobilization. The hydrophobic nature of this matrix produces that the
thermal stability is lower than that obtained immobilizing the enzymes on the more hydrophilic
®
octyl-Sepharose , while the stability in the presence of organic cosolvents is greatly improved using
the support under evaluation.
Regarding the effects on activity, the results are not so clear. Using the same enzyme, for some
®
substrates the activity becomes much higher using MCI GEL CHP20P than that obtained using
octyl-Sepharose®, while using other substrates the situation is the opposite. This difference in activity
is of several orders of magnitude, and so large differences in the substrate specificity has not been
reported to date, and that has been obtained just changing the support, but maintaining the interfacial
activation of the enzyme on the support as the reason for enzyme immobilization. That means that the
only reason for this drastic changes, found for these three enzymes and previously described using
CALB, are based in a different conformation of the immobilized enzyme, whose open form will be
stabilized on a different structure. In fact, each enzyme/support composite has complete different
properties, including response to change in reaction variables (e.g., changes in pH value), behave as
almost fully different biocatalysts.
These changes show the potential of immobilization to tune lipase properties, and the great potential
of this new support to immobilize lipases for many applications. Coupling these results to the high
hydrophobicity of the support, that should reduce the undesired adsorption of water, glycerin, etc, that
are a problem in certain processes [54], this matrix may have indubitable interest as a matrix to
immobilize lipases for many different processes. However, the effects need to be experimentally
determined, as the current technology did not permit to foresee the better matrix to immobilized a
lipase for a determined process [20].
Acknowledgments
We gratefully recognize the support from the Spanish Government, grant CTQ2009-07568 and
CTQ2013-41507-R and CNPq (Brazil). The predoctoral fellowships for García-Galán (Spanish
Government) and dos Santos (CNPq, Brazil) are also recognized. The authors wish to thank Ramiro
Martínez (Novozymes, Spain) for kindly supplying the enzymes used in this research.
The help and comments from Ángel Berenguer (Instituto de Materiales, Universidad de Alicante) are
kindly acknowledged.
Author Contributions
C. Garcia-Galan, Karel Hernandez, Oveimar Barbosa and C.S. dos Santos performed the
experimental work, Rafael C. Rodrigues and Roberto Fernandez-Lafuente designed the experiments
and supervised the work.
Conflicts of Interest
The authors declare no conflict of interest.

Molecules 2014, 19
7642
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©
2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
(