Versatility of divinylsulfone supports permits the tuning of CALB properties during its immobilization

Article abstract of DOI:10.1039/c5ra03798k

The lipase B from C. antarctica (CALB) has been immobilized on divinylsulfone (DVS) activated agarose beads under different conditions (pH 5-10). In the presence of 0.3% Triton X-100, the immobilization rate was rapid at pH 10 and the slowest one was at pH 5. Incubation at pH 10 for 72 h of the immobilized enzymes before blocking of the support with ethylenediamine permitted improvement of the enzyme stability. Enzyme features (activity, stability, specificity versus different substrates, effect of the pH on enzyme properties) were quite different on the different CALB preparations, suggesting the different orientation of the enzyme. The alkaline incubation produced an increase in enzyme activity with some substrates, and some of the DVS-CALB preparations exhibited a higher specific activity than the octyl-preparations. The indirect fluorescence spectrum of the different immobilized preparations confirmed that different structures of the CALB molecules were generated after immobilization.

Full text of DOI:10.1039/c5ra03798k

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Versatility of divinylsulfone supports permits the
tuning of CALB properties during its immobilization
Cite this: RSC Adv., 2015, 5, 35801
ab
ac
d
d
Jose C. S. dos Santos, Nazzoly Rueda, Alfredo Sanchez, Reynaldo Villalonga,
b
a
Luciana R. B. Gonçalves and Roberto Fernandez-Lafuente*
The lipase B from C. antarctica (CALB) has been immobilized on divinylsulfone (DVS) activated agarose
beads under different conditions (pH 5–10). In the presence of 0.3% Triton X-100, the immobilization
rate was rapid at pH 10 and the slowest one was at pH 5. Incubation at pH 10 for 72 h of the
immobilized enzymes before blocking of the support with ethylenediamine permitted improvement of
the enzyme stability. Enzyme features (activity, stability, specificity versus different substrates, effect of
the pH on enzyme properties) were quite different on the different CALB preparations, suggesting the
different orientation of the enzyme. The alkaline incubation produced an increase in enzyme activity with
some substrates, and some of the DVS-CALB preparations exhibited a higher specific activity than the
octyl-preparations. The indirect fluorescence spectrum of the different immobilized preparations
confirmed that different structures of the CALB molecules were generated after immobilization.
Received 3rd March 2015
Accepted 7th April 2015
DOI: 10.1039/c5ra03798k
www.rsc.org/advances
adsorbed to their natural substrates (drops of oils) or any other
1
. Introduction
4,29,30
hydrophobic surface, becoming stabilized.
Enzyme immobilization is a prerequisite for most industrial
their wide substrate specicity, high stability under a broad processes, as a way to easily recover and reuse these relatively
1–6
Lipases are the most utilized enzymes in biocatalysis due to
31–36
range of conditions and reaction media (aqueous, organic expensive biocatalysts and to avoid product contamination.
7
–11
solvent, neoteric solvents)
and broad range of reactions (e.g., Thus, the coupling of enzyme immobilization to the improve-
hydrolysis, esterications, aminations, acydolysis, trans- ment of other enzyme features seems to be a very adequate goal
esterications, and also other promiscuous reactions like in biocatalyst design, and in fact it has been reported
1,6
12–14
perhydrolysis or C–C bond synthesis)
catalyze.
that they are able to improvement in enzyme stability, activity, selectivity, etc. upon
22,23,37–40
immobilization.
The tuning of lipase catalytic properties via immobilization
Moreover, lipase properties, including selectivity, specicity
and activity are very easily modulated by almost any change in is based on involving different regions of the enzyme on the
the enzyme or in the reaction media (including genetic interaction with the support and on the control of the support–
1
5,16
17
22–24
manipulation,
modication of the enzyme surface by polymers or small nano-environments on the enzyme surroundings, may distort
reagents,
medium engineering, physico-chemical enzyme interaction degree;
this may generate different
18–20
21–24
or via immobilization
). This is due to the the regions involved in the immobilization, or may just avoid

exibility of their active center, which is a consequence of the some movements during the opening/closing conformational
conformational changes that the lipases suffer during catalysis, changes. This has been achieved by using different immobili-
involving the movement of an oligopeptide chain (lid or at) zation protocols, which involve different enzyme moieties in the
2
2–24
that usually isolates the active center of lipases from the immobilization.
medium.
However, in some cases a versatile support
25–28
The open form of the lipases becomes strongly may permit to immobilize an enzyme by different orientations
24
by controlling the immobilization conditions. This is the case
of heterofunctional supports, such as glutaraldehyde. This
support has been used to give 4 different preparations of a
lipase just by altering the ionic strength or adding detergents
during immobilization.
Divinylsulfone activated supports have been used for the
a
Departamento de Biocat a´ lisis, Instituto de Cat a´ lisis-CSIC, ICP-CSIC, C/ Marie Curie
2
, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain. E-mail: r@icp.csic.es
41,42
b
Departamento de Engenharia Qu ´ı mica, Universidade Federal do Cear a´ , Campus do
Pici, CEP 60455-760, Fortaleza, CE, Brazil
43–51
c
successful immobilization of some proteins.
Recently, acti-
Escuela de Qu ´ı mica, Grupo de Investigaci ´o n en Bioqu ´ı mica y Microbiolog ´ı a (GIBIM),
Universidad Industrial de Santander, Edicio Camilo Torres 210, Bucaramanga, vated divinylsulfone agarose beads have been described as a
Colombia
suitable support to stabilize enzymes via multipoint covalent
attachment. The reactive group is very stable in a broad range
d
52
Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of
Madrid, 28040 Madrid, Spain
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of pH values (from 5 to 10), capable of reacting with primary and sodium phosphate at pH 6–8 and sodium borate at pH 9–10. At
ꢀ
secondary amines, hydroxyl, phenyl, thiol and imidazol 25 C, all the preparations remained fully active aer incubation
52
groups. However, the reactivity of each enzyme group versus for several hours at any of these pH values.
the vinylsulfone support differed greatly, and also was greatly
5
2
inuenced by the pH value. At pH 10, the Lys residues are only
slightly less reactive that Cys or His (the most reactive ones),
while at pH 5 event he reactivity of the Tyr overpassed the
2
.3. Immobilization of CALB on octyl Sepharose beads
Lipase CALB was immobilized on octyl Sepharose beads at low
ionic strength as previously described. A volume of 1.6 mL of
56
5
2
reactivity of Lys residues. Thus, altering the immobilization
ꢁ
1
commercial enzyme (containing 6.9 mg mL of protein) was
diluted in 88.4 mL of 5 mM sodium phosphate at pH 7, main-
taining a 1/10 support–enzyme solution ratio, (w/v) for 60 min.
Suspension and supernatant samples were withdrawn for evalu-
ation of immobilization through enzymatic activity measurement
as described above. This immobilization strategy also permitted
conditions, it is possible to immobilize an enzyme via different
52
orientations on supports activated with divinylsulfone. The
further long time incubation at alkaline pH value permitted to
increase the number of enzyme–support linkages, increasing
52
the enzyme rigidity.
In this paper, we show the results obtained in the immobi-
lization under different conditions of the most popular lipase,
the lipase B from Candida antarctica,
activated with divinylsulfone with the objective of checking the
possibility of using the features of this support to alter the Immobilization of CALB on CNBr-agarose beads was performed
56
the purication of lipases from contaminant esterases.
53,54
on agarose beads
2.4. Immobilization of CALB on CNBr-agarose beads
57
catalytic properties of lipases. To this goal, the hydrolytic following a protocol previously described for this enzyme.
A
activity versus structurally different substrates and the stability volume of 1 mL of commercial CALB was diluted in 99 mL of 5
of the immobilized enzyme under different conditions will be mM sodium phosphate at pH 7. Then, 6 g of wet CNBr support
ꢀ
studied. Finally, we will try to correlate the changes in enzyme was added. Aer 90 min at 4 C under stirring at 250 rpm,
function aer immobilization on the same support but around 56% of lipase became immobilized on the support. The
following different protocols with changes in the lipase struc- enzyme–support reaction was ended by incubating the support
ture for the rst time in the literature.
with 1 M ethanolamine at pH 8 for 2 h. Finally, the immobilized
preparation was washed with abundant distilled water.
2
. Materials and methods
2
.5. Immobilization of CALB on DVS-agarose beads
.5.1. Preparation of DVS-agarose beads. 1.5 mL divinyl-
sulfone was added to 40 mL of 333 mM sodium carbonate at pH
2.1. Materials
2
Lipase B from Candida antarctica (CALB) was kindly donated by
Novozymes (Spain), p-nitrophenyl butyrate (p-NPB), divinylsul-
fone (DVS), Triton X-100, ethylenediamine (EDA), 8-anilino-1-
naphthalenesulfonic acid (ANS), 2-mercaptoethanol, methyl
mandelate, methyl phenylacetate and ethyl hexanoate were
from Sigma Chemical Co. (St. Louis, MO, USA). Octyl sepharose
beads 4BCL and cyanogen bromide Sepharose beads 4BCL
1
2
2.5 and stirred until the mixture becomes homogeneous, then
g of agarose beads was added and le under gentle agitation
52
for 35 minutes. Finally, the activated support was washed with
an excess of distilled water and stored at 4 C.
ꢀ
2.5.2. Immobilization of CALB on DVS-agarose beads. A 10
g portion of support was suspended in 100 mL of solutions of
(CNBr) were from GE Healthcare. All reagents and solvents were
ꢁ1
ꢀ
CALB (maximum protein concentration was 1 mg mL ) at 25 C
using 10 mM of different buffers (sodium acetate at pH 5, sodium
phosphate at pH 7 or sodium carbonate at pH 10). In some
instances, Triton X-100 was added. In some cases, the immobi-
lized lipase preparations were ltered and a portion of the
derivatives was incubated in 100 mL of 100 mM bicarbonate at
of analytical grade.
All experiments were performed by triplicate and the results
are reported as the mean of this value and the standard devia-
tion (usually under 10%).
ꢀ
pH 10.0 and 25 C for 72 h. As an enzyme–support reaction end-
2.2. Standard determination of enzyme activity
point, all the immobilized biocatalysts were incubated in 1 M
EDA at pH 10 and 25 C for 24 h to block the remaining reactive
This assay was performed by measuring the increase in absor-
bance at 348 nm produced by the released p-nitrophenol in the
hydrolysis of 0.4 mM p-NPB in 50 mM sodium phosphate at pH
ꢀ
groups on the support (this was the optimal blocking reagent
52
using chymotrypsin and this support). Finally, the immobilized
preparation was washed with an excess of distilled water and
ꢀ
ꢁ1
ꢁ1
7
.0 and 25 C (3 ¼ 5150 M cm under these conditions). To
start the reaction, 50–100 mL of lipase solution or suspension
ꢀ
stored at 4 C.
were added to 2.50 mL of substrate solution. One unit of activity
(
U) was dened as the amount of enzyme that hydrolyzes 1 mmol
2
.6. Thermal inactivation of different CALB immobilized
of p-NPB per minute under the conditions previously described.
Protein concentration was determined using Bradford's
method, bovine serum albumin was used as the reference.
preparations
55
To check the stability of the different enzyme derivatives, 1 g of
In the studies of the effects of pH on the enzyme activity, the immobilized enzyme was suspended in 5 mL of 50 mM sodium
protocol was similar but the buffer in the measurements was acetate at pH 5, sodium phosphate at pH 7 or sodium carbonate
changed according to the pH value: sodium acetate at pH 5, at pH 9 and at different temperatures. Periodically, samples were
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withdrawn and the activity was measured using pNPB. Half-lives buffer was sodium acetate at pH 5, sodium phosphate at pH 7
were calculated from the observed inactivation courses.
and sodium bicarbonate at pH 8.5. All experiments were carried
out at 25 C under continuous stirring. The conversion degree
ꢀ
was analyzed by RP-HPLC (Spectra Physic SP 100 coupled with
an UV detector Spectra Physic SP 8450) using a Kromasil C18 (15
cm ꢂ 0.46 cm) column. Samples (20 mL) were injected and
2
.7. Stability assays in the presence of dioxane
Enzyme preparations were incubated in mixtures of 70%
dioxane/30% 100 mM Tris buffer at pH 7 and at different
temperatures to proceed with their inactivation. Periodically,
samples were withdrawn and the activity was measured using p-
NPB as described above. Half-lives were calculated from the
observed inactivation courses. The acetonitrile presented in the
measurement samples had no signicant effect on enzyme
activity determination experiments.
ꢁ
1
eluted at a ow 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 time of 3.4 minutes while the
ester has a retention time of 14.2 minutes. One unit of enzyme
activity was dened as the amount of enzyme necessary to
produce 1 mmol of hexanoic acid per minute under the condi-
tions described above. Activity was determined by triplicate with
a maximum conversion of 20–30%, and data are given as
2
.8. Hydrolysis of methyl mandelate
2
5
00 mg of the immobilized preparations was added to 2 mL of average values.
0 mM substrate in 100 mM sodium acetate at pH 5, 100 mM
sodium phosphate at pH 7 or 100 mM sodium carbonate at pH
2
.11. Fluorescence studies of the different immobilized
ꢀ
8.5 and 25 C under continuous stirring. The conversion degree
enzyme preparations
was analyzed by RP-HPLC (Spectra Physic SP 100 coupled with
an UV detector Spectra Physic SP 8450) using a Kromasil C18 (15
cm ꢂ 0.46 cm) column. Samples (20 mL) were injected and
The immobilized enzyme preparations (150 mg) were mixed
with 15 mL of 13.5 mM 8-anilino-1-naphthalenesulfonic acid
(ANS) solution in 10 mM Tris–HCl buffer, pH 7.0. The mixtures
ꢁ
1
eluted at a ow 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
time of 2.5 minutes while the ester has a retention time of 10
minutes. One unit of enzyme activity was dened as the amount
of enzyme necessary to produce 1 mmol of mandelic acid per
minute under the conditions described above. Activity was
ꢀ
were incubated at 25 C during 1 h under magnetic stirring. The
samples were centrifuged and the emission uorescence
spectra of the supernatant solutions were recorded aer exci-
tation at 360 nm by using a Cary Eclipse Spectrophotometer
58
(Varian).
determined by triplicate with a conversion ranging 20–30%, and 3. Results
data are given as average values.
3.1. Immobilization of CALB on divinylsulfone activated
agarose beads at different pH values
2
.9. Hydrolysis of methyl phenylacetate
Fig. 1 shows the immobilization courses of CALB at pH 5, 7 and
2
5
00 mg of the immobilized preparations were added to 2 mL of
10. It should be remarked that free CALB remained fully active
3
mM substrate in 100 mM buffer containing 50% CH CN. The
under all assayed conditions (not shown results). Surprisingly,
the immobilization was very rapid in all cases, even though at
pH 5 the reactivity of most nucleophilic groups of a protein
buffers were sodium acetate at pH 5, sodium phosphate at pH 7
and sodium bicarbonate at pH 8.5. All experiments were carried
out at 25 C under continuous stirring. The conversion degrees
ꢀ
52
versus vinylsulfone should be quite reduced. Furthermore, an
were analyzed by RP-HPLC (Spectra PhysicSP 100 coupled with
an UV detector Spectra Physic SP 8450) using a Kromasil C18 (15
cm ꢂ 0.46 cm) column. Samples (20 mL) were injected and
ꢁ
1
eluted at a ow rate of 1.0 mL min using a mixture of aceto-
nitrile: 10 mM ammonium acetate aqueous solution (35 : 65,
v/v) and pH 2.8, as mobile phase and UV detection was per-
formed at 230 nm. The acid has a retention time of 3 minutes
while the ester has a retention time of 12 minutes. One unit of
enzyme activity was dened as the amount of enzyme necessary
to produce 1 mmol of phenyl acetic acid per minute under the
conditions described above. The activity was determined by
triplicate with a maximum conversion of 20–30%, and data are
given as average values.
Fig. 1 Immobilization courses of CALB at pH 5, 7 and 10 on DVS-
agarose. Experimental conditions are detailed in Section 2. Circles,
solid black line: suspension pH 5; circles, solid dash line: supernatant
pH 5; square, solid black line: suspension pH 7; square, dash line:
supernatant pH 7; triangles, solid black line: pH10 suspension; trian-
2
.10. Hydrolysis of ethyl hexanoate
Enzyme activity was determined by using ethyl hexanoate; 200
mg of the immobilized preparations were added to 2 mL of 25
mM substrate in 50 mM buffer containing 50% CH
3
CN. The gles, solid dash line: supernatant pH 10.
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Fig. 2 Structure of the activated support.
increase in enzyme activity aer immobilization was appreci- incubated at pH 12) DVS support at pH 5 or 7 were incubated in
ated, approximately 50% in the 3 cases. the presence of detergent just aer immobilization, more than
These facts could be explained if the enzyme was immobi- 80% of the enzyme released from the support. When this
lized via another mechanism, such as physical adsorption. This experiment was performed on the preparation at pH 10, less
could be, for example, the interfacial activation of the lipase in than 10% of the immobilized enzyme was released, showing
the fairly hydrophobic divinylsulfone layer on the agarose that most of the enzyme was covalently attached to the support
56
surface. This hydrophobicity feature of the support was not (although it is not clear which one is the rst step of the
52
detected using chymotrypsin. Fig. 2 shows the structure of the immobilization; covalent attachment or interfacial activation;
45,51
activating group.
This group is moderately hydrophobic, so at least a 10% of the enzyme molecules is not covalently
that a dense layer of this group may enable interfacial activation immobilized aer 3 hours but it is already immobilized).
56
of the enzyme. To check if any physical adsorption could be
Thus, a new batch of CALB immobilizations on DVS-agarose
the cause of the immobilization of CALB, the reactive groups in was carried out at pH 5, 7 and 10, but in the presence of enough
the support were blocked by incubation with 2-mercaptoetha- detergent to prevent lipase adsorption on the inactive DVS
ꢀ
52
nol or destroyed by incubation at pH 12 and 50 C. These support (0.3% Triton X-100) (Fig. 4). Immobilization was rela-
unreactive supports were incubated in the presence of CALB tively rapid at pH 10 (full immobilization aer 3 h). At pH 7,
and even though the effects on enzyme activity were not iden- immobilization was slower (70% aer 24 h) and even slower still
tical, the immobilization rates remained pH independent and at pH 5 (under 30% aer 24 h). These results tted better with
were very similar to those of the activated support (results not the expected chemical reactivity of the enzyme groups at
52
shown). Aer these treatments, it has been described that different pH values versus the DVS activated support.
aminoacids cannot immobilize on the support, because their
chemical reactivity has been destroyed, and the immobilization
of the enzyme conrmed that the covalent attachment was not
the rst step in the immobilization of CALB on DVS activated
agarose in the previous experiments.
3.2. Effect of Triton X-100 on the immobilization of CALB on
divinylsulfone support beads
A detergent is able to desorb the enzyme from a hydrophobic
support, even a very hydrophobic one, and may be used to
59,60
prevent the lipase immobilization via interfacial activation.
By progressively adding Triton X-100 to the DVS-support and the
lipase suspension, it was possible to reduce the adsorption of
the enzyme on the inactivated support (Fig. 3). Using 0.3% Fig. 3 Effect of Trit o´ n X-100 on the immobilization of CALB on
inactivated DVS-supports. The support was incubated 24 h in 0.1 M
NaOH to destroy the vinylsulfone groups. Experimental conditions are
detailed in Section 2. Circles, solid black line: supernatant without
Trit o´ n X-100; squares, solid black line: supernatant with 0.05% Trit o´ n
detergent, CALB did not immobilize on any of the inactivated
supports. These results conrmed that the immobilization on
this support could be founded on the interfacial activation of
CALB on the fairly hydrophobic surface of the support. In fact, if
X-100; triangles, solid black line: supernatant with 0.15% Trit o´ n X-100;
the enzymes adsorbed on the reactive (neither blocked nor rhombus, solid black line: supernatant with 0.3% Trit o´ n X-100.
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Table 1 Activities of the different CALB preparations versus p-NPB.
DVS-CALB was blocked using EDA. Activity was determined at pH 7
ꢀ
and 25 C as indicated in Section 2. Activity is given in mmol of substrate
hydrolyzed per minute and mg of immobilized enzyme. The prepa-
ration of the biocatalyst is in Section 2
Biocatalysts
Activity
DVS-pH 5-EDA
DVS-pH 5/pH 10-EDA
DVS-pH 7-EDA
DVS-pH 7/pH 10-EDA
DVS-pH 10 (2 h) EDA
DVS-pH 10 (72 h)-EDA
Octyl
7.79 ꢃ 1.7
22.3 ꢃ 2.2
20.44 ꢃ 2.9
27.15 ꢃ 2.5
23.79 ꢃ 1.91
32.15 ꢃ 1.94
16.92 ꢃ 2.16
5.90 ꢃ 1.17
Fig. 4 Immobilization courses of CALB on DVS-supports in presence
of 0.3% Triton at pH 5, 7 and 10. Experimental conditions are detailed in
Section 2. Circles, solid black line: suspension pH5; circles, solid dash
line: supernatant pH 5; square, solid black line: suspension pH 7;
square, dash line: supernatant pH 7; triangles, solid black line: pH 10
suspension; triangles, solid dash line: supernatant pH 10.
CNBr
The enzyme immobilized at pH 7 suffered an increase in the
activity during the alkaline incubation (around 220%), and this
effect was even more relevant if the enzyme had been immo-
bilized at pH 5 (over 250%). The most active preparations were
those incubated at pH 10 in all cases (Table 1), even though
under these conditions a higher enzyme–support chemical
reaction should occur. This increase in enzyme activity upon
incubation at alkaline pH values could be explained as a func-
tion of enzyme distortions caused by the enzyme–support
reaction that, in this case, presented positive effects on enzyme
activity.
In order to compare the enzyme properties aer immobili-
zation, CALB was also immobilized on octyl agarose and CNBr
agarose. The enzyme immobilized on octyl agarose (results not
shown) presented less than 60% of the activity of the enzyme
immobilized on DVS support and incubated at pH 10. It should
be considered that the small lid of CALB makes that the activity
of the enzyme is not signicantly increased aer immobiliza-
tion on octyl agarose (around a 10%). The enzyme immobilized
on CNBr agarose did not signicantly alter its activity (Table 1).
Looking at the activity, the immobilization at pH 10
produced an increase in enzyme activity (around 30%) while at
the other pH values, the activity slightly decreased aer
immobilization. This higher activity at pH 10 is curious, as it
may not be due to a lower intensity of the enzyme–support
52
reaction.
To enhance immobilization yields, a ratio of 1 g of support to
mL of enzyme suspension was used. Under these conditions
3
CALB immobilization was almost complete even at pH 5 aer 24
h (results not shown).
3.3. Effect of the long term incubation at alkaline pH value
on enzyme activity
Aer immobilization, and in order to favor the multipoint
covalent immobilization, the three immobilized CALB bio-
catalysts (immobilized at pH 5, 7 or 10) were incubated at pH 10
for 72 h, aer washing the detergent. Results are shown in
Fig. 5.
3.4. Characterization of the immobilized biocatalysts
The preparation immobilized at pH 10 increased the activity
for 48 h, and later kept that value constant (near 170%).
The 6 new covalent preparations have been compared against
each other and also with the two standard immobilization
protocols, CALB immobilized on CNBr- and octyl-Sepharose.
3.4.1. Activity/pH versus pNPB. Table 1 shows the activities
of the 8 preparations under standard conditions aer blockage.
The hyperactivation caused by the alkaline incubation at pH 10
is clearly visualized, the enzyme immobilized at pH 5 started
with 2 fold less activity than the enzyme immobilized at pH 10,
but aer alkaline incubation, the higher increase on enzyme
activity permitted to almost equilibrate the observed activities.
All of them (except the enzyme just immobilized at pH 5) are
more active than the octyl preparation, which is also slightly
more active than the CNBr preparation.
Fig. 6 shows the enzyme activity/pH prole using the
different immobilized samples. The main difference is found
when comparing the enzymes immobilized on different
supports. The enzyme immobilized on CNBr-Sepharose pre-
sented the maximum of activity at pH 7, with a sharp decrease at
either alkaline or acidic pH values (activity was around 40% at
Fig. 5 Effect of the long term incubation at pH 10 value on enzyme
activity on CALB immobilized on DVS agarose at different pH values:
Experimental conditions are detailed in Section 2. Circles, solid black
line: pH 5; square, solid black line: pH 7; triangles, solid black line:
pH 10.
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multipoint covalent attachment, becoming more stable than
the CNBr-CALB in all cases.
If the inactivation was performed at pH 5, the alkaline
incubation increased the half live from 4.5 to 35 minutes for the
enzyme immobilized at pH 5, if the immobilization was per-
formed at pH 7, the stability increased to a lower extent, from 33
to 60 minutes. The value of the half live of the enzyme immo-
bilized at pH 10 went from 32 to 46 minutes aer the long term
incubation, a value lower than that obtained with the immo-
bilization at pH 7 and incubated at pH 10.
The pattern was somehow similar looking at the inactiva-
tions carried out at pH 7, the enzyme immobilized at pH 7 and
incubated at alkaline conditions was the most stable one, fol-
lowed by the enzyme immobilized at pH 10 and the enzyme
immobilized at pH 5.
Fig. 6 Effect of the pH on the activity versus pNPB of the different
CALB preparations. Experimental conditions are detailed in Section 2.
Circles, solid black line: pH 5; gray circles, solid gray line: pH 5–10;
squares, solid black line: pH 7; grays squares, solid gray line: pH 7–10;
triangles, solid black line: pH10; gray triangles, solid gray line: pH 10–
1
0. stars, solid black line: octyl; gray stars, solid gray line: CNBr.
At pH 9, the situation varied. The enzyme immobilized at pH
7
presented a stability similar to that of the octyl, and the
pH 5 and 10). Using octyl agarose as support, the maximum alkaline incubation of this preparation permitted to double the
activity was found at pH 8, and the decrease in activity at acidic half-life. The stabilities of the enzymes immobilized at pH 5 or
and alkaline pH values is milder (55% at pH 5 and 70% at pH 10 were quite similar, both aer immobilization and aer long
1
0). The enzyme immobilized on DVS support under different term alkaline incubation before blocking. In both cases, the
conditions presented the maximum activity at the highest pH stability became similar to that of the octyl-CALB aer the
used in the study (pH 10), and only slight differences were alkaline incubation. It may be likely that at pH 9 the cause of the
found on the immobilization pH or long term incubation at inactivation is a conformational change in another area of the
alkaline pH value. The enzyme immobilized at pH 5 showed an enzyme or just a chemical modication of some groups, this
1
8% or 25% of the maximum activity at pH 5, for the non- can explain the signicant qualitative change in the stability of
incubated or long term incubated enzyme preparations the different preparations.
respectively. Both enzyme preparations immobilized at pH 7
Considering that in all cases the support was the same for
exhibited 40% of the maximum activity at pH 5, while the the DVS immobilized enzymes, and that the long term incu-
preparations immobilized at pH 10 showed around 30% of this bation of 3 days should permit a similar reaction between the
activity.
.4.2. Thermal stability under different conditions at must be related to populations of enzyme molecules having
different pH values. Table 2 shows the half-lives of the different different orientations, with different relevance for enzyme
enzyme and the support, the differences on enzyme stability
3
61,62
CALB preparations under different inactivation conditions. We stability
or different density of groups able to react with the
only show the results obtained in the temperature where the support, giving differences in the nal intensity of the multi-
22
inactivations have a rate that permitted to obtain reliable results point covalent attachment.
in a reasonable time.
The high thermostability of the lipases adsorbed on hydro-
63
The most stable preparation was that obtained using octyl- phobic supports has been previously described. These prepa-
agarose when the inactivations were performed at pH 5 or 7. rations are much more stable than the glyoxyl agarose-CALB,
The just immobilized DVS preparations were far less stable, but and this was explained by the very stable conformation that the
64
their stabilities improved aer long-term incubation to favor open form of the adsorbed lipases presented, and the
moderate amount of nucleophilic groups that many lipases
Table 2 Half-lives (expressed in minutes) of the different CALB preparation under different inactivation conditions. Experiments were performed
as described in Section 2. * The enzyme retained full activity during the inactivation assay
Inactivation conditions
ꢀ
ꢀ
ꢀ
ꢀ
CALB preparation
pH 5, 55 C
pH 7, 55 C
pH 9, 55 C
70% dioxane, 25 C, pH 7
DVS-pH 5-EDA
DVS-pH 5–pH 10-EDA
DVS-pH 7-EDA
VS-pH 7–pH 10-EDA
DVS-pH 10-EDA
DVS-pH 10–pH 10-EDA
Octyl
4.5 ꢃ 0.3
35 ꢃ 1.2
33 ꢃ 1.0
60 ꢃ 2.4
32 ꢃ 1.3
46 ꢃ 2.2
240 (100%)*
45 ꢃ 3.3
3 ꢃ 0.3
4.5 ꢃ 0.3
33 ꢃ 1.9
27 ꢃ 2.1
60 ꢃ 3.2
4.2 ꢃ 0.4
25 ꢃ 1.2
30 ꢃ 2.1
4.6 ꢃ 0.3
5 ꢃ 0.3
5.3 ꢃ 0.7
1.5 ꢃ 0.2
7.3 ꢃ 0.3
1.7 ꢃ 0.2
2.6 ꢃ 0.4
0.17 ꢃ 0.02
0.21 ꢃ 0.02
10 ꢃ 1.1
33 ꢃ 2.2
60 ꢃ 3.3
4 ꢃ 0.2
25 ꢃ 1.2
240 (100%)*
24 ꢃ 2.3
CNBr
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presented in its surface make complex a very intense multipoint
Using ethyl hexanoate, results are quite diverse depending
covalent attachment (e.g., CALB has 9 Lys plus the Leu 1, all of on the biocatalyst. The highest activity was usually found at pH
65,57
them exposed to the medium).
5, except for the preparation immobilized at pH 5 and then
3.4.3. Solvent stability. In opposition to the results incubated at alkaline pH, where the maximum activity was
obtained during thermal inactivations, Table 2 shows that in all found at pH 7. The enzyme just immobilized at pH 7 on DVS
cases the DVS preparations were by far more stable that the octyl was the most active one at pH 5 and pH 7 while at pH 8.5 the
or CNBr-Sepharose immobilized enzymes when they were most active one was the octyl-Sepharose preparation. The long
incubated in the presence of 70% dioxane. Analyzing the DVS term incubation at alkaline pH of the DVS preparations usually
preparations blocked just aer immobilization, the most stable decreased the enzyme activity, mainly at pH 5. The enzyme
biocatalyst was that prepared at pH 5 (half live of 5 minutes), immobilized at pH 5 is the one with the most drastic change
being the stability of the enzymes immobilized at pH 7 and 10 aer alkaline incubation, with a shi in the maximum activity
very similar (1.5–1.7 minutes). However, aer the long term at pH 7 (becoming more active than the enzyme just immobi-
incubation the enzyme immobilized at pH 7 greatly improved lized at pH 5 under these conditions, that is, alkaline incuba-
the stability (to more than 7 minutes), while the enzyme tion produced an hyper-activation at pH 7). On the other hand,
immobilized at pH 5 maintained its stability practically unal- the enzyme immobilized at pH 5 and at pH 10 improved the
tered aer alkaline incubation and the enzyme immobilized at activity aer alkaline incubation if the activity was determined
pH 10 improved its the stability by only 50%.
at pH 8.5. In general, the effect of the change of the pH in the
The low stability of CALB immobilized on octyl-agarose in activity determination presented a more drastic effect on DVS
the presence of dioxane may be related to the enzyme desorp- preparations without long term alkaline incubation (e.g., from
ꢁ
1
ꢁ1
tion caused by the presence of this very high cosolvent 425 U mg to 24 U mg using the enzyme immobilized at pH
concentration, the free enzyme is rapidly inactivated under 10) than in octyl or CNBr preparations (activity at pH 8.5 was
52,66
these drastic conditions.
around 60% and 30% than that at pH 5, respectively). Long term
The different stability of the enzymes immobilized at incubation at alkaline pH reduced this effect of the pH on DVS-
different pH value on DVS activated supports, where aer long CALB activity.
term alkaline incubation the only difference may be the enzyme
Using methyl phenylacetate, at pH 5 the most active prepa-
orientation, suggests that the inactivation of CALB follows a rations are two DVS preparations, those just immobilized at pH
ꢁ
1
ꢁ1
different route on different inactivation conditions, some 7 (22.5 U mg ) and pH 10 (18.7 U mg ). At pH 8.5, octyl and
protein regions are more relevant on the stability at certain CNBr CALB preparations presented the highest activity, while at
conditions, while some other areas may be more relevant on pH 7 the most active preparations were CNBr and DVS immo-
61,62,67
other experimental conditions.
bilized at pH 7. The lowest activity for all preparations immo-
3.4.4. Activity versus different esters. Immobilization has bilized on DVS was that found at pH 8.5, except for the enzyme
been reported to alter enzyme specicity and the inuence of immobilized at pH 5 on DVS and submitted to alkaline incu-
the pH on the activity, if enzyme orientation on the support or bation that have the minimum activity at pH 7. The highest
22,24
the intensity of the enzyme–support interaction is different.
activity depended on the immobilization protocol. The DVS
Thus, differences in enzyme specicity or inuence on activity/ preparations immobilized at pH 5 had a clear maximum at pH
pH curve upon different immobilization protocols can reinforce 5; while both preparations immobilized at the other two pH
the idea on a different enzyme orientation on the support values have not a clear maximum (similar activities are detected
surface. Three different substrates have been used at 3 different at pH 5 and 7). Octyl and CNBr CALB had a clear maximum at
pH values: esters formed by an aliphatic acid (ethyl hexanoate), pH 7. Long term alkaline incubation decreased enzyme activity
one aromatic acid (phenylacetate) or one aromatic and chiral in all cases, but the intensity of this effect depended on the
one (mandelic acid) and the results are resumed on Table 3.
immobilization pH and activity determination pH.
Table 3 Activity of different CALB preparations versus different substrates at different pH values. Experimental details may be found in Section 2.
MM, methyl mandelate; MPA, methyl phenylacetate; EH, ethyl hexanoate. The activity is given in mmol of substrate hydrolyzed per minute and mg
of immobilized enzyme
CALB
preparations
MM/pH 5
MM/pH 7
MM/pH 8.5 MPA/pH 5
MPA/pH 7
MPA/pH 8.5 EH/pH 5
EH/pH 7
EH/pH 8.5
Octyl
CNBr
DVS-pH 5
DVS-pH 5–pH 10
DVS-pH 7
DVS-pH 7–pH 10
DVS-pH 10
DVS-pH 10–pH 10
16.45 ꢃ 0.8
55.00 ꢃ 2.8 41.07 ꢃ 2.1 14.02 ꢃ 0.7 24.27 ꢃ 1.2 19.17 ꢃ 1
450.00 ꢃ 23 300.00 ꢃ 15 273.44 ꢃ 14
28.25 ꢃ 1.1 124.15 ꢃ 5.0 85.61 ꢃ 3.4 15.32 ꢃ 0.6 30.54 ꢃ 1.2 19.00 ꢃ 0.8 627.85 ꢃ 25 436.65 ꢃ 17 197.44 ꢃ 8
11.61 ꢃ 0.6
39.6 ꢃ 0.9
52.17 ꢃ 2.6
56.13 ꢃ 2
58.78 ꢃ 2.9 23.28 ꢃ 1.2
82.32 ꢃ 1.1 35.08 ꢃ 1.8
6.97 ꢃ 0.3
2.86 ꢃ 0.1
3.94 ꢃ 0.2
1.38 ꢃ 0.1
2.39 ꢃ 0.1 200.89 ꢃ 10 139.18 ꢃ 7
1.95 ꢃ 0.1 74.40 ꢃ 4 194.20 ꢃ 10
30.97 ꢃ 2
50.22 ꢃ 3
57.07 ꢃ 2.9 52.41 ꢃ 2.6 22.47 ꢃ 1.1 25.64 ꢃ 1.3
5.43 ꢃ 0.3 760.87 ꢃ 38 456.52 ꢃ 23 188.52 ꢃ 9
78.80 ꢃ 3.9 86.43 ꢃ 1.9
67.92 ꢃ 3.5 51.07 ꢃ 2.6 18.65 ꢃ 0.9 17.59 ꢃ 0.9
69.00 ꢃ 3.5 29.11 ꢃ 1.5 6.47 ꢃ 0.3 6.68 ꢃ 0.3
9.34 ꢃ 0.5
9.29 ꢃ 0.5
3.36 ꢃ 0.2 217.39 ꢃ 11 157.07 ꢃ 8
8.13 ꢃ 0.4 425.00 ꢃ 21 191.25 ꢃ 10
142.66 ꢃ 7
24.38 ꢃ 1
38.53 ꢃ 2
12.50 ꢃ 0.6
8.35 ꢃ 0.4
2.71 ꢃ 0.1
62.50 ꢃ 3
41.25 ꢃ 2
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Using mandelic ester, new changes were found. The most
active preparations at pH 5 were both preparations immobilized
at pH 7 on DVS, at pH 7 the most active preparations were the
CNBr preparation and the enzyme immobilized at pH 5 or pH 7
and long term submitted to alkaline incubation before block-
ing. At pH 8.5, the most active biocatalysts were those immo-
bilized at pH 7 and long term incubated and the CNBr
preparation. There are examples where the highest activity was
found at pH 7 (octyl, CNBr, both DVS immobilized at pH 5 and
both immobilized at pH 10). The enzyme immobilized at pH 7
has not a clear maximum activity, and aer incubation this
optimum is clearly at pH 8.5. The long term incubation of the
DVS preparations used to have a positive effect on enzyme
activity, except when the enzyme was immobilized at pH 10,
where the alkaline incubation decreased the enzyme activity
when measured at pH 5 or 8.5, while having almost no effect at
pH 7.
Fig. 7 Spectra of ANS incubated in the presence of different DVS
immobilized CALB. Experimental conditions are detailed in Section 2.
Line (a) blocked DVS-support; line (b) DVS-CALB-pH 5, line (c) DVS-
CALB pH 5 + 72 h at pH 10, line (d) DVS-CALB pH 10; line (e) DVS-CALB
pH 10 + 72 h at pH 10.
Thus, CALB immobilized following different protocols on
DVS-activated supports (different immobilization pH values,
long term incubation or not under alkaline conditions) pre-
sented very different enzyme specicity and very different
response to changes on environmental conditions, conrming
that the different preparations have different orientation and/or
When the enzyme is immobilized at pH 10 (line d), the effect
of the further alkaline incubation is in the opposite direction
68
degree of enzyme–support interaction.
.4.5. Evaluation of the structure of different CALB
3
(line e), the uorescence signal increased aer the alkaline
immobilized preparations. The inuence of the different
immobilization strategies on the 3D conformation of the
enzyme was determined by using the ANS-binding uorescence
assays. ANS is a hydrophobic uorescent dye that strongly binds
the clusters from hydrophobic amino acid side chains in b-
incubation, less hydrophobic groups are partially exposed sug-
gesting a more rigid and compact structure. Again, the changes
in enzyme properties could be correlated to conformational
changes.
Moreover, it is clear that the difference in the exposition of
protein hydrophobic groups of the enzyme immobilized at pH 5
and that immobilized at pH 10, in both cases aer 72 h of
incubation at pH 10 before support blocking is quite signicant,
with much higher exposition using the enzyme immobilized at
pH 5 and incubated at pH 10. The results may be explained by
the implication of different areas of the enzyme in the multi-
point covalent attachment. This produced fully different effects
on the enzyme structure (making more compact one and more
relaxed the other), The effects on the exposition of the hydro-
phobic groups surrounding the active of the lipase (the small lid
and adjacent areas) may be also considered. These differences
may explain the drastic changes of enzyme properties when
immobilized at different pH values discussed along this paper,
and suggest that the areas reacting with the support for those 72
h could be different.
59
sheet conformations of proteins. Usually, a great density of
those hydrophobic clusters is well protected from the solvent in
native enzymes due to the rigid packing of the globular protein
conformation. Accordingly, a decrease in the uorescence
intensity of the ANS dye can be attributed to its binding to the
exposed hydrophobic regions in partially unfolded proteins.
Fig. 7 shows the uorescence emission spectra of the bio-
catalysts prepared through different immobilization protocols.
In comparison with the raw support (line a), the uorescence
intensity of ANS decreased aer incubation with all immobi-
lized lipase preparations. This fact can be ascribed to the
presence of the enzyme molecules on the support surface, and
thus, to the binding of ANS molecules to the exposed hydro-
phobic clusters in these proteins. On the other hand, the results
obtained using the biocatalyst prepared by immobilization at
pH 5 and further incubation at 10 (line c) showed the lowest

uorescence signal, much lower than using the enzyme
immobilized at pH 5 (line b). This result suggested that the
immobilization approach based on two consecutive incubation
4. Conclusions
steps at pH 5 and 10 leads to protein conformations with Immobilization of CALB on DVS-supports under different
partially exposed hydrophobic b-sheet clusters, and accord- conditions permits to have covalently immobilized prepara-
ingly, more prone to bind the hydrophobic ASN molecules, than tions exhibiting very different properties. The change in the
when the enzyme is just immobilized at pH 5 and them blocked. immobilization pH permits to alter the enzyme specicity,
That is, alkaline incubation produced conformational changes activity and stability, while further incubation under alkaline
on the enzyme that led to the exposition of more hydrophobic conditions (described as a way to improve the enzyme support
52
groups to the medium.
reaction) also produced changes in enzyme features. The
indirect determination of the ANS incubated enzyme
35808 | RSC Adv., 2015, 5, 35801–35810
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
uorescence showed that the different enzyme derivatives have 19 C. Garcia-Galan, J. C. S. dos Santos, O. Barbosa, R. Torres,
different structures.
Thus, DVS activated supports may be a potent way to tuning
lipase properties via immobilization. The DVS activation of
E. B. Pereira, V. C. Corberan, L. R. B. Gonçalves,
R. Fernandez-Lafuente and L. R. B. Goncalves, Process
Biochem., 2014, 49, 604–616.
supports compatible with organic media may increase the range 20 J. C. S. dos Santos, C. Garcia-Galan, R. C. Rodrigues, H. B. de
of reactions where the biocatalysts may be used and provide
new data on the different behavior of CALB immobilized on
different supports.
Sant'Ana, L. R. B. Gonçalves and R. Fernandez-Lafuente,
Enzyme Microb. Technol., 2014, 60, 1–8.
´
21 C. Garcia-Galan, A. Berenguer-Murcia, R. Fernandez-
Lafuente and R. C. Rodrigues, Adv. Synth. Catal., 2011, 353,
2
885–2904.
Acknowledgements
2
2 K. Hernandez and R. Fernandez-Lafuente, Enzyme Microb.
We gratefully recognize the support from the MINECO of
Technol., 2011, 48, 107–122.
´
Spanish Government, by the grant CTQ2013-41507-R. The 23 R. C. Rodrigues, C. Ortiz, A. Berenguer-Murcia, R. Torres and
predoctoral fellowships for Ms Rueda (Colciencias, Colombian
R. Fern ´a ndez-Lafuente, Chem. Soc. Rev., 2013, 42, 6290–6307.
Goberment) and Mr dos Santos (CNPq, Brazil) are also recog- 24 O. Barbosa, R. Torres, C. Ortiz, A. Berenguer-Murcia,
nized. The authors wish to thank Mr Ramiro Mart ´ı nez (Novo-
R.
C.
Rodrigues
and
R.
Fernandez-Lafuente,
zymes, Spain) for kindly supplying the enzymes used in this
Biomacromolecules, 2013, 14, 2433–2462.
´
research. The help and comments from Dr Angel Berenguer 25 A. M. Brzozowski, U. Derewenda, Z. S. Derewenda,
(
Instituto de Materiales, Universidad de Alicante) are kindly
G. G. Dodson, D. M. Lawson, J. P. Turkenburg,
F. Bjorkling, B. Huge-Jensen, S. A. Patkar and L. Thim,
Nature, 1991, 351, 491–494.
acknowledged.
2
6 K. K. Kim, H. K. Song, D. H. Shin, K. Y. Hwang and S. W. Suh,
Structure, 1997, 5, 173–185.
References
1
2
3
K. E. Jaeger and T. Eggert, Curr. Opin. Biotechnol., 2002, 13, 27 K. E. Jaeger, S. Ransac, H. B. Koch, F. Ferrato and
90–397. B. W. Dijkstra, FEBS Lett., 1993, 332, 143–149.
R. Sharma, Y. Chisti and U. C. Banerjee, Biotechnol. Adv., 28 M. Cygler and J. D. Schrag, Biochim. Biophys. Acta, 1999,
001, 19, 627–662.
1441, 205–214.
K. E. Jaeger and M. T. Reetz, Trends Biotechnol., 1998, 16, 29 R. Verger, Trends Biotechnol., 1997, 15, 32–38.
3
2
3
96–403.
30 C. Cambillau, S. Longhi, A. Nicolas and C. Martinez, Curr.
Opin. Struct. Biol., 1996, 6, 449–455.
4
5
R. D. Schmid and R. Verger, Analysis, 1998, 37, 1608–1633.
A. Pandey, S. Benjamin, C. R. Soccol, P. Nigam, N. Krieger 31 I. Chibata, T. Tosa and T. Sato, J. Mol. Catal., 1986, 37, 1–24.
and V. T. Soccol, Biotechnol. Appl. Biochem., 1999, 29(pt 2), 32 W. Hartmeier, Trends Biotechnol., 1985, 3, 149–153.
119–131.
33 E. Katchalski-Katzir, Trends Biotechnol., 1993, 11, 471–478.
6
J. Zhang, H. Shi, D. Wu, Z. Xing, A. Zhang, Y. Yang and Q. Li, 34 J. F. Kennedy, E. H. M. Melo and K. Jumel, Chem. Eng. Prog.,
Process Biochem., 2014, 49, 797–806.
1990, 86, 81–89.
7
8
P. Adlercreutz, Chem. Soc. Rev., 2013, 42, 6406–6436.
35 A. M. Klibanov, Science, 1983, 219, 722–727.
P. Lozano, J. M. Bernal, G. S ´a nchez-G ´o mez, G. L ´o pez-L ´o pez 36 D. Brady and J. Jordaan, Biotechnol. Lett., 2009, 31, 1639–
and M. Vaultier, Energy Environ. Sci., 2013, 6, 1328. 1650.
P. Lozano, J. M. Bernal and A. Navarro, Green Chem., 2012, 37 R. Fernandez-Lafuente, Enzyme Microb. Technol., 2009, 45,
4, 3026. 405–418.
0 P. Lozano, J. M. Bernal and M. Vaultier, Fuel, 2011, 90, 3461– 38 C. Mateo, J. M. Palomo, G. Fernandez-Lorente, J. M. Guisan
9
1
1
3
467.
and R. Fernandez-Lafuente, Enzyme Microb. Technol., 2007,
40, 1451–1463.
1
1
1 Y. Fan and J. Qian, J. Mol. Catal. B: Enzym., 2010, 66, 1–7.
2 C. Li, X.-W. Feng, N. Wang, Y.-J. Zhou and X.-Q. Yu, Green 39 M. Petkar, A. Lali, P. Caimi and M. Daminati, J. Mol. Catal. B:
Chem., 2008, 10, 616. Enzym., 2006, 39, 83–90.
3 E. Busto, V. Gotor-Fern ´a ndez and V. Gotor, Chem. Soc. Rev., 40 U. Guzik, K. Hupert-Kocurek and D. Wojcieszy n´ ska,
010, 39, 4504–4523. Molecules, 2014, 19, 8995–9018.
4 M. Kapoor and M. N. Gupta, Process Biochem., 2012, 47, 555– 41 O. Barbosa, R. Torres, C. Ortiz and R. Fernandez-Lafuente,
69. Process Biochem., 2012, 47, 1220–1227.
1
1
2
5
´
1
1
5 N. J. Turner, Nat. Chem. Biol., 2009, 5, 567–573.
6 U. T. Bornscheuer and M. Pohl, Curr. Opin. Chem. Biol., 2001,
42 O. Barbosa, C. Ortiz, A. Berenguer-Murcia, R. Torres,
R. C. Rodrigues and R. Fernandez-Lafuente, RSC Adv.,
2014, 4, 1583.
5
, 137–143.
1
7 J. M. Palomo, G. Fernandez-Lorente, C. Mateo, C. Ortiz, 43 F. J. Lopez-Jaramillo, M. Ortega-Mun ˜o z, A. Megia-Fernandez,
R. Fernandez-Lafuente and J. M. Guisan, Enzyme Microb.
Technol., 2002, 31, 775–783.
F. Hernandez-Mateo and F. Santoyo-Gonzalez, Bioconjugate
Chem., 2012, 23, 846–855.
´
1
8 R. C. Rodrigues, A. Berenguer-Murcia and R. Fernandez- 44 P. Prikryl, J. Lenfeld, D. Horak, M. Ticha and Z. Kucerova,
Lafuente, Adv. Synth. Catal., 2011, 353, 2216–2238. Appl. Biochem. Biotechnol., 2012, 168, 295–305.
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 35801–35810 | 35809

View Article Online
RSC Advances
Paper
4
4
4
5 M. Ortega-Mu n˜ oz, J. Morales-Sanfrutos, A. Megia-Fernandez, 57 O. Barbosa, M. Ruiz, C. Ortiz, M. Fern ´a ndez, R. Torres and
F. J. Lopez-Jaramillo, F. Hernandez-Mateo and F. Santoyo-
Gonzalez, J. Mater. Chem., 2010, 20, 7189.
6 J. C. Begara-Morales, F. J. L ´o pez-Jaramillo, B. S ´a nchez-Calvo,
A. Carreras, M. Ortega-Mu n˜ oz, F. Santoyo-Gonz ´a lez,
R. Fernandez-Lafuente, Process Biochem., 2012, 47, 867–876.
58 G. V Semisotnov, N. A. Rodionova, O. I. Razgulyaev,
V. N. Uversky, A. F. Gripas' and R. I. Gilmanshin,
Biopolymers, 1991, 31, 119–128.
F. J. Corpas and J. B. Barroso, BMC Plant Biol., 2013, 13, 61. 59 K. Hernandez, C. Garcia-Galan and R. Fernandez-Lafuente,
7 A. L. Medina-Castillo, J. Morales-Sanfrutos, A. Megia- Enzyme Microb. Technol., 2011, 49, 72–78.
Fernandez, J. F. Fernandez-Sanchez, F. Santoyo-Gonzalez 60 C. Garcia-Galan, O. Barbosa, K. Hernandez, J. C. S. dos
and A. Fernandez-Gutierrez, J. Polym. Sci., Part A: Polym.
Chem., 2012, 50, 3944–3953.
Santos, R. C. Rodrigues and R. Fernandez-Lafuente,
Molecules, 2014, 19, 7629–7645.
4
4
5
8 K. Labus, A. Turek, J. Liesiene and J. Bryjak, Biochem. Eng. J., 61 J. Mansfeld and R. Ulbrich-Hofmann, Biotechnol. Appl.
011, 56, 232–240. Biochem., 2000, 32(pt 3), 189–195.
9 J. Bryjak, J. Liesiene and B. N. Kolarz, Colloids Surf., B, 2008, 62 J. Mansfeld, G. Vriend, B. Van Den Burg, V. G. H. Eijsink and
1, 66–74. R. Ulbrich-Hofmann, Biochemistry, 1999, 38, 8240–8245.
0 M. D. Bale Oenick, S. J. Danielson, J. L. Daiss, 63 J. M. Palomo, G. Mu n˜ oz, G. Fern ´a ndez-Lorente, C. Mateo,
2
6
M. W. Sunderberg and R. C. Sutton, Ann. Biol. Clin., 1990,
8, 651–654.
R. Fern ´a ndez-Lafuente and J. M. Guis ´a n, J. Mol. Catal. B:
Enzym., 2002, 19–20, 279–286.
4
5
1 J. Morales-Sanfrutos, J. Lopez-Jaramillo, M. Ortega-Mu n˜ oz, 64 G. H. Peters, O. H. Olsen, A. Svendsen and R. C. Wade,
A. Megia-Fernandez, F. Perez-Balderas, F. Hernandez- Biophys. J., 1996, 71, 119–129.
Mateo and F. Santoyo-Gonzalez, Org. Biomol. Chem., 2010, 65 M. Galvis, O. Barbosa, M. Ruiz, J. Cruz, C. Ortiz, R. Torres
8
, 667–675.
and R. Fernandez-Lafuente, Process Biochem., 2012, 47,
2373–2378.
5
2 J. C. S. dos Santos, N. Rueda, O. Barbosa, J. F. Fern ´a ndez-
S ´a nchez, A. L. Medina-Castillo, T. Ram ´o n-M ´a rquez, 66 G. Fernandez-Lorente, M. Filice, D. Lopez-Vela, C. Pizarro,
M. C. Arias-Martos, M. C. Mill ´a n-Linares, J. Pedroche,
M. del, M. Yust, L. R. B. Gonçalves and R. Fernandez-
Lafuente, RSC Adv., 2015, 5, 20639–20649.
3 E. M. Anderson, K. M. Larsson and O. Kirk, Biocatal.
Biotransform., 1998, 16, 181–204.
L. Wilson, L. Betancor, Y. Avila and J. M. Guisan, J. Am. Oil
Chem. Soc., 2010, 88, 801–807.
67 V. Graz u´ , F. L ´o pez-Gallego, T. Montes, O. Abian, R. Gonz ´a lez,
J. A. Hermoso, J. L. Garc ´ı a, C. Mateo and J. M. Guis ´a n, Process
Biochem., 2010, 45, 390–398.
5
5
4 V. Gotor-Fern ´a ndez, E. Busto and V. Gotor, Adv. Synth. Catal., 68 J. M. Palomo, G. Fern ´a ndez-Lorente, C. Mateo, M. Fuentes,
2
006, 348, 797–812.
R. Fern ´a ndez-Lafuente and J. M. Guisan, Tetrahedron:
Asymmetry, 2002, 13, 1337–1345.
5
5 M. M. Bradford, Anal. Biochem., 1976, 72, 248–254.
5
6 R. Fernandez-Lafuente, P. Armis ´e n, P. Sabuquillo,
G. Fern ´a ndez-Lorente and J. M. Guis ´a n, Chem. Phys. Lipids,
1998, 93, 185–197.
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