Easy stabilization of interfacially activated lipases using heterofunctional divinyl sulfone activated-octyl agarose beads. Modulation of the immobilized enzymes by altering their nanoenvironment

Article abstract of DOI:10.1016/j.procbio.2016.04.002

Octyl-agarose is a support that permits the one step immobilization, stabilization and purification of lipases. However, the enzyme may be released from the support under drastic conditions. This paper describes a new heterofunctional support, octyl agarose beads activated with divinyl sulfone, that has proved to be useful to produce very stable and active biocatalysts of lipases from Candida rugosa (CRL), Rhizomucor miehei (RML) and Thermomyces lanuginosus (TLL), able to work under any reaction conditions without risking enzyme desorption. The three enzymes failed in immobilization on glyoxyl-octyl supports for different reasons. The immobilization at pH 5 permitted to keep the good properties of octyl agarose. Further incubation at pH 8 permitted to establish at least one covalent enzyme-support bond per enzyme molecule (preventing the risk of enzyme desorption), avoiding the inactivation produced at pH 10, and the final result is that all three new biocatalysts are more active than the octyl-glyoxyl counterparts and much more stable (e.g.; 20 using CRL). The end of the enzyme-support reaction was achieved via blocking the vinylsulfone groups with different nucleophiles (cationic, anionic, hydrophobic, etc). This not only determined the final enzyme stability, but also the activity, selectivity and even specificity of the different immobilized preparations.

Full text of DOI:10.1016/j.procbio.2016.04.002

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Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Easy stabilization of interfacially activated lipases using
heterofunctional divinyl sulfone activated-octyl agarose beads.
Modulation of the immobilized enzymes by altering their
nanoenvironment
Tiago L.de Albuquerquea,b,1, Nazzoly Ruedaa,c,1, Jose C.S. dos Santosa,d
,
Oveimar Barbosae, Claudia Ortizf, Baris Binayg, Ece Özdemira,h, Luciana R.B. Gonc¸ alvesb,
Roberto Fernandez-Lafuentea,∗
a Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM-CSIC Madrid, Spain
b Departamento de Engenharia Química, Universidade Federal do Ceará, Campus do Pici, CEP 60455-760 Fortaleza, CE, Brazil
c Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Universidad Industrial de Santander, Edificio Camilo Torres 210,
Bucaramanga, Colombia
d Instituto de Engenharias e Desenvolvimento Sustentável, Universidade da Integrac¸ ão Internacional da Lusofonia Afro-Brasileira, CEP 62785-000 Acarape,
CE, Brazil
e Departamento de Química, Facultad de Ciencias. Universidad del Tolima, Ibagué, Colombia
f
Escuela de Microbiología, Universidad Industrial de Santander, Bucaramanga, Colombia
g Istanbul Arel University, Faculty of Science and Letters, Department of Molecular Biology and Genetics, Tepekent, Buyukcekmece, Istanbul, Turkey
h Yıldız Technical University, Graduate School of Natural and Applied Sciences, Department of Chemistry. Bes¸iktas¸ Merkez Yerles¸kesi, Bes¸iktas¸, Istanbul,
Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Octyl-agarose is a support that permits the one step immobilization, stabilization and purification of
lipases. However, the enzyme may be released from the support under drastic conditions. This paper
describes a new heterofunctional support, octyl agarose beads activated with divinyl sulfone, that has
proved to be useful to produce very stable and active biocatalysts of lipases from Candida rugosa (CRL),
Rhizomucor miehei (RML) and Thermomyces lanuginosus (TLL), able to work under any reaction conditions
without risking enzyme desorption. The three enzymes failed in immobilization on glyoxyl-octyl supports
for different reasons. The immobilization at pH 5 permitted to keep the good properties of octyl agarose.
Further incubation at pH 8 permitted to establish at least one covalent enzyme-support bond per enzyme
molecule (preventing the risk of enzyme desorption), avoiding the inactivation produced at pH 10, and
the final result is that all three new biocatalysts are more active than the octyl-glyoxyl counterparts and
much more stable (e.g., 20 using CRL). The end of the enzyme-support reaction was achieved via blocking
the vinylsulfone groups with different nucleophiles (cationic, anionic, hydrophobic, etc). This not only
determined the final enzyme stability, but also the activity, selectivity and even specificity of the different
immobilized preparations.
Received 3 March 2016
Received in revised form 22 March 2016
Accepted 1 April 2016
Available online xxx
Keywords:
Lipase interfacial activation
Heterofunctional support
Divinyl sulfone
Enzyme hyperactivation
Enzyme stabilization
Covalent immobilization
© 2016 Elsevier Ltd. All rights reserved.
1. Introduction
uses to reuse this expensive catalyst or improve some enzyme prop-
erties [6–11]. Immobilization may be specially interesting if it may
Lipases are among the most used biocatalysts [1–5]. These very
robust enzymes require their immobilization for most industrial
be coupled to purification, another requirement for some uses of
lipases [12].
Recently, the immobilization of lipases on heterofunctional
octyl-glyoxyl supports has been revealed as a potent tool to reach
one step immobilization, purification, hyperactivation and strong
stabilization of the lipases[13–18]. It permitted to specifically
immobilize the open form of the lipases on the support permit-
ting to get a significant increment in activity after immobilization
[19]. Moreover, this stabilized open form of the lipase has been
∗
Corresponding author at: Instituto de Catálisis-CSIC, C/Marie Curie 2, Campus
UAM-CSIC, Cantoblanco, 28049 Madrid, Spain.
E-mail addresses: rfl@icp.csic.es, rfernandezlafuente@hotmail.com
(R. Fernandez-Lafuente).
1
Both authors have evenly contributed to this paper.
http://dx.doi.org/10.1016/j.procbio.2016.04.002
1359-5113/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: T.L.de Albuquerque, et al., Easy stabilization of interfacially activated lipases using heterofunctional
divinyl sulfone activated-octyl agarose beads. Modulation of the immobilized enzymes by altering their nanoenvironment, Process
Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.04.002

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described to be much more stable than the lipase form in open-
ing/closing equilibrium [20,21]. In fact, lipases immobilized on
octyl-agarose have proved to be much more stable than enzyme
immobilized via multipoint covalent attachment, overpassing the
stability of enzymes immobilized on glyoxyl or vinylsulfone acti-
vated supports[22,23]. The use of octyl-glyoxyl is based on a first
immobilization at neutral pH value of the lipases via interfacial
activation over the octyl layer of the support [19,24] and later,
the incubation at pH 10 increased the reactivity of the ␧-amino
group of Lys and permitted the establishment of covalent link-
ages between the adsorbed lipases and the glyoxyl groups on the
support [18]. The immobilization is first via interfacial activation
because the glyoxyl groups are unable to immobilize standard pro-
teins at neutral pH values [25]. This strategy improved the stability
of some enzymes keeping the advantages of the octyl supports,
permitting even at least a partial reactivation of the inactivated
enzymes via unfolding/refolding strategies [15,26]. These biocat-
alysts were more stable than the just octyl preparations, that
was previously stated to be among the most stable preparations
described in literature, even more than covalently attached lipases
preparations [22,24,27]. Although this immobilization method was
very useful, it presented several limitations. The enzyme needs to
be stable at alkaline pH value to get the enzyme-support reac-
tion [18], and that did not occur with all lipases (e.g., lipase
from Rhizomucor miehei lost more than 50% of the activity during
alkaline incubation) [18]. Moreover, some lipases have very few
primary amino groups in the face of the active center, and in some
instances a significant percentage of enzyme molecules is not cova-
lently attached to the support (e.g., more than 30% of the lipase
molecules from Thermomyces lanuginosus immobilized on octyl-
glyoxyl released from the OCGLX support when incubated in the
presence of detergents)[18] This has been solved by the chemi-
cal amination of the lipases, which has provided a simple way to
increase the reactivity towards the glyoxyl groups of the support
[28]. However, this strategy introduced an additional step in the
biocatalyst preparation although may improve enzyme properties
in certain cases[29].
incubated at neutral or alkaline pH value to increase the enzyme-
support reactivity, but it is not necessary to use pH 10 [22,30,39,27]
like using octyl-glyoxyl [15,18]. The vinyl sulfone group has sev-
eral advantages regarding the glyoxyl group for getting at least one
enzyme-support covalent attachment: it may involve many differ-
ent groups on the enzyme surface in the immobilization (Lys, but
also His, Cys or Tyr), the attachment is irreversible and may be
achieved under a broad range of pH values[30]. Additionally, the
spacer arm is longer, reducing the steric hindrances for the cova-
lent enzyme-support reaction caused by the layer of octyl groups
when compared to the glyoxyl groups[30,39]. In fact, it has been
shown that the DVS activated agarose enable a much more intense
multipoint covalent attachment than the glyoxyl groups[30,39].
The possibilities using this kind of supports go further. After
immobilization, and as a reaction end point, the remaining vinyl
sulfone groups may be blocked using different nucleophiles[23,30].
This blocking step (using pure epoxy or DVS activated supports) has
been used as an additional tool to tailor enzyme properties[23,40].
Using lipases immobilized on supports activated by DVS, it has been
shown that the blocking step permits to alter the lipase proper-
ties, not only stability, but also activity, selectivity or specificity[23].
This was explained by different interactions between the enzyme
and the support, which could promote some conformational lipase
changes. The conformational changes of lipases make that their
active center is quite flexible making relatively easy modulate
the final properties (even if in an empiric way) via different tools
[41–47]. Using OCDVS supports, the presence of a certain amount of
blocking reagents (in a moderate spacer arm) with different phys-
ical properties may induce some additional interactions with the
already immobilized lipase.
In this paper, we show the results obtained with three differ-
ent lipases after the optimization of the immobilization protocol
on octyl-DVS supports: the lipase from Candida rugosa (CRL)[48],
R. miehei (RML)[49,50] and T. lanuginosus (TLL)[51]. These three
lipases have some problems using octyl-glyoxyl (enzyme inactiva-
tion or moderate covalent immobilization yield)[15,18]. Moreover,
the immobilization on agarose activated with DVS in the case of
the lipase from R. miehei did not permitted to obtain an active
enzyme preparation, while using TLL the activity recovery was
satisfactory but the stability was far below that of the octyl-TLL
preparations[27]. Here, we intend to evaluate if the new hetero-
functional OCDVS support is able to solve these problems.
In this new paper, we have prepared a new type of hetero-
functional support, octyl activated with divinyl sulfone (OCDVS) to
introduce vinyl sulfone groups on the support surface. The vinyl-
sulfone group may be easily introduced by modification of the
hydroxyl groups of octyl-agarose with DVS [30], and this group may
react with primary and secondary amino, imidazole, thiol, pheno-
lic groups, etc. The use of this group to immobilize proteins was
first proposed by Porath [31], but this activation protocol has been
scarcely utilized [32–38].
2. Materials and methods
2.1. Materials
Recently, the advantages of this group in enzyme immobi-
lization have been studied[22,30,39]. The vinylsulfone moiety is
very stable (permitting long-term immobilization), it is able to
react with very different moieties (primary and secondary amino
groups but also hydroxyl, imidazole or thiol groups [30] These make
that these supports may produce a rapid and intense multipoint
covalent attachment of enzymes[30,39]. Moreover, it has been
described that this support may immobilize proteins at a pH range
from 5 to 10, involving different residues in the first immobiliza-
tion depending on the immobilization pH value[30,39]. However,
immobilization at acid pH value on this support is very slow, far
slower than at neutral or alkaline pH value [30]. This is critical for
our strategy, as we intended that the first immobilization of the
enzyme on the support still was via interfacial activation versus the
acyl-layer. Considering the rapid immobilization of lipases on octyl-
agarose at any pH value [19], this new heterofunctional support
may permit the first lipase immobilization via interfacial activa-
tion if the immobilization is performed at acidic pH. Later, this
immobilized enzyme may get a covalent attachment with the sup-
port. To accelerate this reaction, the immobilized enzyme may be
Octyl-Sepharose beads matrix (4BCL) was from GE Healthcare.
Solutions of lipase from T. lanuginosus (TLL) (36 mg of protein/mL)
and lipase from R. miehei (RML) (13.7 mg of protein/mL) were kindly
supplied by Novozymes (Spain). Lipase from C. rugosa (CRL) (in
powder form, 40% protein (w/w)), divinyl sulfone (DVS), R and S
methyl mandelate, methyl phenyl acetate, ethyl hexanoate and p-
nitrophenyl butyrate (p-NPB) were obtained from Sigma Chemical
Co. (St.Louis, MO, USA). All reagents and solvents were of analyti-
cal grade. The preparation of octyl glyoxyl-agarose (OC-GLX) was
performed as previously described [18].
2.2. Standard determination of enzyme activity
All experiments were performed by triplicate and the results
are reported as the mean of this value and the standard deviation
(under 10%).
This reaction was followed by recording the increase in
absorbance at 348 nm produced by the released p-nitrophenol in
Please cite this article in press as: T.L.de Albuquerque, et al., Easy stabilization of interfacially activated lipases using heterofunctional
divinyl sulfone activated-octyl agarose beads. Modulation of the immobilized enzymes by altering their nanoenvironment, Process
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Fig. 1. Effect of the incubation pH on the stability of different OCDVS-preparations. The Figure shows Thermal inactivation courses of the different enzymes immobilized in
OCDVS and incubated at different pH values for 24 h. Experiments have been performed as described in Methods. Rhombus: incubated at pH 9, Stars: incubated at pH 8.5,
triangles: incubated at pH 8, circles: incubated at pH 7, squares: incubated pH 5. Experiments were performed in 50 mM buffer acetate at pH 5 and at 55 ◦C for RML (Panel
A), 60 ◦C for CRL (Panel B) and 70 ◦C for TLL (Panel C).
Fig. 2. Effect of the incubation time on the stability of different OCDVS-preparations. The Figure shows the thermal inactivation courses of the different enzymes immobilized
in OC-DVS incubated at pH 8 for different times and blocked with glycine. Experiments have been carried out as described in Methods. Rhombus: 2 h, stars: 6 h, triangles:
24 h, circles: 72 h, squares: 168 h, pointed line: OC. Experiments were performed at pH 5 in buffer acetate 50 mM at 55 ◦C for RML (Panel A), 60 ◦C for CRL (Panel B) and 70 ◦
for TLL (Panel C).
C
the hydrolysis of 0.4 mM p-NPB in 25 mM sodium phosphate at pH
7.0 and 25 ◦C (⑀ = 5150 M−1cm−1 under these conditions). To ini-
tialize the reaction, 50–200 ␮L of lipase solution or suspension was
added to 2.5 mL of substrate solution. The amount of enzyme that
hydrolyzes 1 ␮mol of p-NPB per min under the conditions described
previously is defined as one activity unit. Protein concentration
was determined using Bradfordı´s method [52] using bovine serum
albumin as reference.
phosphate at pH 12.5, to a final concentration of 0.35 M of DVS, to
get a homogeneous solution. Then 2 g of octyl-agarose beads were
added and left under gentle agitation for 35 min. The final support
was filtered and washed with distilled water and stored at 4 ◦C.
2.4. Immobilization of enzymes on OC, OCGLX and OCDVS
supports
A mass of 10 g of support was suspended in 100 mL of enzy-
matic solutions (containing 100 p-NPB units) of enzyme at 25 ◦C in
5 mM sodium acetate at pH 5. The activities of both supernatants
and suspensions were followed using p-NPB. After immobilization
the suspensions were filtered and washed with distilled water.
The immobilized enzymes on OCDVS were in some instances re-
2.3. Preparation of octyl-divinyl sulfone agarose support (OCDVS)
The support was adapted from
a previously described
procedure[30], in which agarose was substituted by octyl-agarose.
A volume of 1.5 mL of DVS was stirred in 40 mL of 333 mM sodium
Please cite this article in press as: T.L.de Albuquerque, et al., Easy stabilization of interfacially activated lipases using heterofunctional
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Fig. 3. Effect of the blocking reagent on the stability of OCDVS-lipase preparations at pH 5. The Figure shows the thermal inactivation courses of the different enzymes
immobilized in OC-DVS incubated at pH 8 for 6 h and blocked with different reagents. Other details are in Methods section. Crosses: aspartic acid, rhombus: cysteine, stars:
ethanolamine, triangles: ethylenediamine, circles: glycine, full squares: mercaptoethanol, empty squares: propanethiol. Experiments were performed in 50 mM buffer acetate
at pH 5 and 55 ◦C for RML (Panel A), 60 ◦C for CRL (Panel B) and 70 ◦C for TLL (Panel C).
A
B
100
75
50
25
0
100
75
50
25
0
0
2
4
6
0
2
4
6
Time, (h)
Time, (h)
150
C
100
50
0
0
6
12
Time, (h)
18
24
Fig. 4. Comparison of the stability of different lipase preparations in the presence of organic solvents. The Figure shows the inactivation courses of the different enzymes
immobilized in OC-DVS incubated at pH 8 for 6 h and blocked with different reagents. Other details are in Methods section. Triangles: optimized OCDVS; circles: OCGLX;
Dotted line: OC. Experiments were performed at 30 ◦C using acetonitrile 25% for RML (Panel A), acetonitrile 45% for CRL (Panel B) and Dioxane 70% (Panel C).
suspended in 50 mM sodium acetate at pH 5 or sodium phosphate
at pH 7, 8 and 8.5 or sodium carbonate at pH 9 and 25 ◦C for dif-
ferent times, to analyze the effect of the pH on the properties of
the immobilized lipases. When the enzyme was immobilized on
octyl–glyoxyl, the washed biocatalyst was resuspended in sodium
bicarbonate at pH 10.0 for different times, to favor the enzyme-
support covalent reaction and reduced with sodium borohydride
[18].
propanethiol or some aminoacids (cysteine, glycine and aspartic
acid) at pH 8 and 25 ◦C for different times to block the remain-
ing reactive groups. Finally, the biocatalysts were washed with an
excess of distilled water and stored at 4 ◦C.
2.6. Desorption of the enzyme from the supports
To analyze if the enzymes were covalently attached to the
supports, the derivatives were incubated with an appropriate con-
centration of detergent, where all the enzyme activity was released
from the support[18]. This should release the enzyme molecules
just adsorbed via interfacial activation but not covalently attached.
Thus, samples of 1 g of different biocatalyst were suspended at 25 ◦C
in 10 mL of 0.5% and 0.3% of Triton X-100 for CRL and RML, respec-
2.5. Blocking of the OCDVS supports
As
a reaction end point, the enzymes immobilized in
OCDVS were incubated in 1 M of different nucleophiles[27] as
ethanolamine (ETA), ethylenediamine (EDA), mercaptoethanol,
Please cite this article in press as: T.L.de Albuquerque, et al., Easy stabilization of interfacially activated lipases using heterofunctional
divinyl sulfone activated-octyl agarose beads. Modulation of the immobilized enzymes by altering their nanoenvironment, Process
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Table 1
5
tively and 0.7% of CTAB for TLL. A reference suspension, having inert
agarose beads and the same amount of lipase was submitted exactly
to the same treatment, to detect the effects of the detergent on
enzyme activity or stability.
Activity versus p-NPB of different lipase preparations. The activity of OC preparation
is taken as 100%. The numbers between parenthesis is the expressed activity after
washing with detergents (see methods).
Biocatalyst
Relative activity, (%)
OC-RML
100 6 (0)
2.7. Stability of the different lipase biocatalyst
OCGLX-RML
OCDVS-RML
OC-CRL
OCGLX-CRL
OCDVS-CRL
OC-TLL
47 5 (40 3)
115 8 (113 8)
100 7 (0)
35 2 (30 4)
70 3 (72 4)
100 7 (0)
2.7.1. Thermal inactivation of different enzyme immobilized
preparations
1 g of immobilized enzyme was suspended in 5 mL of 50 mM
of sodium acetate at pH 5, sodium phosphate at pH 7 or sodium
bicarbonate at pH 9 at different temperatures. Periodically, samples
were withdrawn and the activity was measured using p-NPB. Half-
lives were calculated from the observed inactivation courses.
OCGLX-TLL
OCDVS-TLL
72 3 (50 5)
90 5 (89 6)
Activity was determined by triplicate with a maximum conversion
of 20–30%, and data are given as average values.
2.7.2. Inactivation of different preparations in the presence of
organic co-solvents
Enzyme preparations were incubated in mixtures of acetonitrile
or 1,4-dioxane/100 mM Tris–HCl pH 7 at 30 ◦C. Periodically, sam-
ples were withdrawn and the activity was measured using p-NPB as
described above. Half-lives were calculated from the observed inac-
tivation courses. The organic co-solvents presented in the samples
did not have a significant effect on enzyme activity during p-NPB
assay (results not shown).
2.8.3. Hydrolysis of methyl phenyl acetate
50 mM solutions of methyl phenyl acetate in 50% CH3CN/50%
buffer 100 mM sodium phosphate at pH 7 were prepared. Reaction
was initialized by adding 200 mg of the enzymatic preparations
to 5 mL of the substrate solution. 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 × 0.46 cm) column. Samples (20 ␮L) were injected and
eluted at a flow rate of 1.0 mL/min using acetonitrile/10 mM ammo-
nium acetate aqueous solution (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 4 min while the ester has a retention time
of 12 min. The enzyme activity was measured in ␮mol of phenyl
acetic acid per minute under the conditions described above. Activ-
ity was determined by triplicate, using a maximum conversion of
20–30%.The data are given as average values.
2.8. Determination of the hydrolytic activity of the biocatalyst
versus different substrates
2.8.1. Hydrolysis of R or S methyl mandelate
50 mM solutions of R or S methyl mandelate in 50 mM sodium
phosphate buffer at pH 7 were prepared. Reactions were started by
adding 1 g of the lipase preparations into 5 mL of the substrate solu-
tion. The suspensions were maintained at 25 ◦C under continuous
stirring. The reactions were followed by RP-HPLC (Spectra Physic
SP 100 coupled with an UV detector Spectra Physic SP 8450) using
a Kromasil C18 (15 cm × 0.46 cm) column. Samples (20 ␮L) were
injected and eluted at a flow rate of 1.5 mL/min using acetoni-
trile/10 mM ammonium acetate (35:65, v/v) at pH 2.8 as mobile
phase and UV detection was performed at 230 nm. The retention
times of the different compounds were: acid: 3 min, ester: 7 min.
The enzyme activity was expressed in ␮mol of mandelic acid per
minute and mg of enzyme under the conditions described above.
Activity was determined by triplicate, using a maximum conversion
of 20–30%. The data are given as average values.
2.9. SDS-PAGE analysis
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was car-
ried out according to [53] using a Miniprotean tetra-cell (Biorad),
12% running gel in a separation zone of 9 cm × 6 cm, and a con-
centration zone of 5% polyacrylamide. One hundred milligrams of
the immobilized enzyme samples were re-suspended in 1 mL of
rupture buffer (2% SDS and 10% mercaptoethanol). Then, this sus-
pension was boiled for 5 min and a 20 ␮L aliquot of the supernatant
was used in the SDS-PAGE analysis. The heating in the presence of
SDS is enough to release all the adsorbed enzyme molecules, while
it cannot break the support-enzyme secondary amino bonds[18].
Gels were stained with Coomassie brilliant blue. Low molecular
weight markers from Fermentas were used (14.4–116 kDa).
2.8.2. Hydrolysis of ethyl hexanoate
25 mM solutions of ethyl hexanoate in 50 mM sodium phos-
phate buffer at pH 7, in some instances containing 25% (V/V)
CH3CN to get a homogenous system instead of a biphasic system,
were prepared. Reactions were initialized by adding 200 mg of the
enzymatic preparations to 5 mL of the substrate solution. All exper-
iments 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 Kro-
masil C18 (15 cm × 0.46 cm) column. Samples (20 ␮L) were injected
and eluted at a flow rate of 1.5 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. When
a substrate/enzyme suspension biphasic system existed, a sample
of 100 ␮L was withdrawn under very vigorous stirring, mixed with
a volume of acetonitrile and filtered before injection in the HPLC.
Hexanoic acid had a retention time of 2.5 min while the ester pre-
sented a retention time of 10 min. One unit of enzyme activity was
defined as the amount of enzyme necessary to produce 1 ␮mol of
hexanoic acid per minute under the conditions described above.
3. Results
3.1. Immobilization of different lipases on OC and OCDVS
At pH 5, it has been shown that the protein immobilization rate
on DVS supports is very low [30], while the immobilization on OC
supports is very rapid at any pH value [19]. Thus, pH 5 was utilized
to immobilize the lipases on the OCDVS support. Fig. 1S shows the
immobilization courses of RML, CRL and TLL on OC and OCDVS sup-
ports. The immobilization courses on both supports are very similar
in all cases, with also very similar increment on enzyme activity
after immobilization (more than 3.5 folds using RML, more than 3
folds using CRL and around 80% using TLL). This means that while
in all cases 10 U of enzyme/g of support was offered, after immo-
Please cite this article in press as: T.L.de Albuquerque, et al., Easy stabilization of interfacially activated lipases using heterofunctional
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bilization on OCDVS, 35, 30 or 18 U/g of support were obtained for
RML, CRL and TLL, respectively.
3.3. Effect of the incubation pH on the stability of OCDVS-lipase
preparations
Thus, the presence of the DVS groups under the layer of octyl
groups has no significant effect either on the immobilization rate
or on the hyperactivation effect caused by the immobilization.
Moreover, if the immobilized enzymes were exposed to detergent
just after immobilization, more than 95% of the enzyme molecules
could be desorbed from the support, confirming that the enzyme
molecules were not immobilized via a reaction with the DVS. Fur-
thermore, adding a sufficient concentration of detergent to prevent
enzyme adsorption, immobilization was almost fully prevented
(results not shown).
Before taking a final decision on the optimal condition for
immobilization, the stabilities of the different biocatalysts were
compared. To this study, after the 24 h incubation, the OCDVS
derivatives were blocked with EDA, a group that was found to
be very suitable using other lipases immobilized on just DVS
supports[22,27]. The blocking pH was 8, because at higher pH val-
ues the enzyme activity decreased due to the low stability of RML or
CRL at these more alkaline pH values. After this blocking step, none
of the preparations released enzyme when incubated with deter-
gent or submitted to SDS-PAGE (results not shown), even those
prepared at pH 5. That is, during incubation at pH 8 in the pres-
ence of the nucleophile, the enzyme and the support continue the
reaction, and some new bonds are formed before the blocking is
complete. That implies that the enzyme-support interaction time is
not strictly controlled and differences on enzyme properties incu-
bated at different times may be minimized due to the reactions
occurred during this blocking step at pH 8.
Fig. 1 shows the thermal inactivation courses of the different
lipase preparations. One common feature is that in all cases the
incubation at pH 8 is the condition that permitted the highest sta-
bilization, in some cases very similar to the values at pH 8.5 and 7,
while in other cases, the stabilization is clearly higher when incu-
bated at pH 8. This could be related to the very mild distortion
caused on the enzyme structure during enzyme-support reaction
at pH 7 and 8 compared to that at pH 8.5 and 9. Considering just
the number of enzyme linkages, using a higher pH value it should
be at least as those obtained at pH 8 or higher [30], the reactiv-
ity of the enzyme-support at pH 8 seems to be enough to provide
an adequate number of enzyme-support bond. Stability is clearly
higher than that obtained after incubation at pH 5, where the num-
ber of enzyme-support bonds should be lower (but we have at least
one covalent bond, because there is not enzyme desorption when
incubated in detergent solution).
3.2. Covalent immobilization of the already adsorbed enzymes on
OCDVS supports
Next, we investigated the best strategy to get OCDVS covalently
immobilized enzyme biocatalysts. The experiments were started
using RML as a model enzyme by the intrinsic difficulties of the
immobilization of this enzyme on DVS activated supports. RML
could not be immobilized on this support in an active form [23].
Thus, samples of the already immobilized and hyperactivated RML-
OCDVS preparations were incubated at different pH values (5, 7, 8,
8.5 and 9). Table 1S shows that after 24 h incubation at pH 8, no
activity could be released from the OCDVS-RML when incubated in
detergent. At pH 7 a very small percentage could be detected in the
supernatant (less than 3%), while at pH 5 almost ¾ of the enzyme
molecules could still be released from the support.
Table 1S shows that CRL immobilized even more rapidly in a
covalent way after adsorption on OCDVS than RML. Even the prepa-
ration at pH 5 after 24 h did not release any enzyme activity when
incubated with detergent. TLL could be successfully immobilized
on DVS supports[23], and using OCDVS, 60% of the offered enzyme
is covalently immobilized after 24 h at pH 5, 85% at pH 7 and almost
100% at pH 8 (Table 1S).
In the case of OCDVS-RML, the differences in stability are rela-
tively minor with the incubation pH, although incubation at pH 8
is the pH that provides the highest stability. Using OCDVS-CRL the
preparation with a clear lower stability is that incubated at pH 7,
followed by that incubated at pH 9 where higher distortions have
been produced on the enzyme. The differences between the other
OCDVS-CRL preparations are not very large. The case of OCDVS-
TLL is the one where the differences are higher when changing the
incubation pH. Stability is very high and similar when they have
been incubated at pH 7 or 8; if the incubation has been performed
at pH 8.5 or 9 the enzyme stability was lower, while it is very poor
if they have been prepared at pH 5.
This way, OCDVS supports solved one of the problems of the
use of OC-glyoxyl supports, they can efficiently immobilized 100%
of the enzyme molecules in a covalent way.
Table 2S shows the activity recovery for the different OCDVS-
lipase preparations when incubated at different pH values. Using
OCDVS-RML, the activity after incubation at pH 5 or 9 are the lowest
ones, while the maximum is obtained at pH 8.5, which is very simi-
lar to the activity of the OC-RML (that is, keeping the improvement
on activity caused by the immobilization). Using OCDVS-CRL, the
recovered activity decreased when using a more alkaline pH value,
although at pH 5 a lower activity value was obtained than at pH 7
(at pH 7 the activity is similar to that of OC-CRL). OCDVS-TLL pre-
sented similar activities recoveries at all pH values, slightly higher
at pH 8.5 (at this pH the activity is similar to that of the OC-TLL). It
has been reported that the immobilization or incubation of TLL and
CALB on DVS activated supports before blocking may produce some
increments on enzyme activity, and this has been related to changes
in the enzyme structure[22,27]. This may explain why some of the
preparations are more active when incubated under more drastic
conditions than when incubated under mild conditions; there are
some hyperactivation of the enzyme during enzyme-support reac-
tion caused by the enzyme distortions provoked. The decrease in
activity of RML and CRL when incubated at alkaline pH values may
be related to the low stability of these enzymes under these condi-
tions [15,18]. Therefore, OCDVS supports are also able to solve the
second problem of OC-glyoxyl supports, they can covalently immo-
bilize the enzymes at lower pH values and provide a higher activity
recovery, almost maintaining intact the hyperactivation obtained
after immobilization on the OC supports.
Thus, pH 8 was selected as optimal pH for the three enzymes
considering the global process management and the activity, sta-
bility and full covalent immobilization for all enzymes.
3.4. Effect of incubation time on the stability of the different
OCDVS lipase preparations
The multipoint reaction between an enzyme and a support may
be complex and require some time to get the maximum degree
[22,27,30,39]. For this reason, samples of the OCDVS-lipases were
incubated at pH 8 and blocked with EDA at different times. Since
during the blocking some further enzyme-support reaction may
occur (see above), the real time of enzyme-support reaction is not
strictly defined by the time before blocking and that may minimize
the impact of this variable. In this study, the OC-lipase derivatives
were included as a reference where there were no covalent link-
ages. Table 3S shows that the enzyme activity decreased when the
incubation time increased, although not very drastically. OCDVS-
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RML incubated between 2 and 168 h decreased its activity only to
73%, OCDVS-CRL to 65% and OCDVS-TLL only loses around 15% of
the activity after 168 h.
At pH 5, using OCDVS-RML, the effect of the blocking reagent
is very high on the stability. The most stable one was that pre-
pared using Gly. Thiolated compounds produced biocatalysts with
greatly decreased stability, while EDA also has a very negative effect
compared to Gly.
The effect of the blocking reagents using OCDVS-CRL is not so
strong, only the use of Cys gave a much more unstable preparation.
Propanethiol and Gly (quite different agents) were the best ones
and permitted to get similar stabilizations, EDA was again one of
the worst blocking reagents, in opposition to the results found with
other enzymes using pure DVS activated supports [22,23,27,30,39].
OCDVS-TLL blocked with Gly was also the most stable prepara-
tion, with Cys and EDA among the worst ones. EDA was found to
be the most adequate using the just DVS activated supports. In this
case, Asp was the second worst and mercaptoethanol and ETA were
the second best.
It is hard to give a clear explanation on these results. It seems
that small and ionic mixed glycine is the best one, although the
ion mixed but longer Cys is among the worst in all cases. Proper
explanations of these results seems to require potent modelling
tool to predict both, the enzyme-supports interactions and the final
results of these interactions on enzyme stability. From a practi-
cal point of view, and considering activity and stability, Gly was
selected as optimal blocking reagent for all OCDVS-lipases for point
3.5.2.1. These blocking almost kept unaltered the hyperactivation
obtained by immobilization on OC supports and provide the highest
stabilizations.
All OCDVS-RML preparations were much more stable than the
OC-preparation (Fig. 2), up to the point that it was not possible to
find a temperature where OC preparations had a reliable half live
and the half live of the OCDVS preparation could be determined
in a reasonable time. This also occurred for the other enzymes.
For RML, maximum stability was obtained at 72 h of incubation,
but also considering the activity and the relatively low increase in
enzyme stability, 6 h seemed like a suitable compromise solution.
Using OCDVS-CRL, the maximum stability was observed after
6 h and the same occurred with TLL (Fig. 2), in agreement with the
hypothesis that a distortion on the OCDVS-lipase structure, near the
layer of octyl groups, was not compensated by a new covalent bond.
Thus, we propose as optimal conditions for these three preparations
pH 8 and 6 h of incubation before the blocking step.
3.5. Effect of the blocking step
Next, we have evaluated the impact on enzyme features of the
use of blocking reagents with different physical (and chemical)
nature. Anionic, cationic or hydrophobic blocking reagents have
been used, to analyze if they can somehow alter the overall effect
of the octyl groups and somehow alter enzyme features. Results are
listed below
3.5.1. Effect on p-NPB activity
3.6. Comparison of enzyme stability with other lipase
immobilized biocatalyst
Table 4S shows the effects of the nature of the blocking reagent
on the lipase activity versus p-NPB. Using OCDVS-RML, and consid-
ering 100% the activity of the unblocked preparation (incubated at
pH 8 for 6 h before blocking), all preparations decreased the activ-
ity during the blocking. This may be due to the promotion of more
enzyme-support linkages before the support is properly blocked,
or really to the new enzyme-support physical interactions with the
new groups. The small ETA is the best blocking reagent, leaving the
enzyme activity almost intact. EDA permitted to keep 80% and Gly
around 85% initial activity, while Asp permitted to retain 75% of the
initial activity. All thiolated reagents produced severe decreases on
enzyme activity, including Cys, suggesting that it may be acting on
the disulfide bonds of this enzyme. In fact, similar deactivations
were found when the OC-RML was incubated under these condi-
tions (results not shown), therefore the effect is a direct effect on
the disulfide bonds and not in the microenvironment.
Using CRL, thiolated compounds again produced drastic drops in
activity, except propanethiol (Table 4S). Gly is the reagent that gave
the highest activity recovery (92%), the other reagents produced
preparations with activity ranging from 76% to 50%.
OCDVS-TLL blocking promoted an increase on enzyme activ-
ity using some of the reagents (Asp, Gly or ETA), in agreement
with previous results using pure DVS-TLL catalysts[22,27]. EDA
and propanethiol produced activities near to 100%, while Cys and
mercaptoethanol produced the lowest activities (42% and 68%,
respectively) (Table 4S).
Next, optimal OCDVS-lipase biocatalyst of each enzyme has
been compared to the respective OC and OCGLX preparations
[15,18], that are among the most stable biocatalysts of these
enzyme reported in literature as previously discussed. Figs. 2–4S
shows the results of thermal inactivations at different pH values.
The new OCDVS-lipase preparations not only were much more sta-
ble than the octyl preparation, but were also much more stable than
the OCGLX preparations under all assayed inactivation conditions.
Table 1 shows that the described stabilization was coupled to a
twofold increase in the activity of the new biocatalysts compared
to OCGLX for CRL and RML. Using TLL the differences in activity
were shorter, but after washing with CTAB the TLL preparations;
the differences reached again around 2 folds. OCGLX-TLL released
30% of the activity while OCDVS-TLL did not release any activity.
Fig. 4 shows that in organic solvents the new biocatalysts are also
much more stable than the OC or OCGLX preparations.
Thus, this protocol enables to have much better stability than
the OC preparations, with relative low decrease of activity caused
by the whole protocol that permitted to keep the hyperactiva-
tion achieved by immobilization on octyl supports. Considering the
effect of time on enzyme stability (to prolong the incubation time
from 2 to 6 h improve enzyme stability, while at 2 h no enzyme
was released to the medium), the difference with the stability of
OCGLX preparations must be found on a higher number of enzyme-
supports bonds using OCDVS supports. This higher usefulness of
DVS activated support versus OCGLX to produce an intense multi-
point covalent attachment between enzyme and support has been
already shown using pure DVS supports[22,30,39].
It is curious that Cys seems to be among the worst blocking
reagents for all enzymes, when mercaptoethanol is more adequate
to break disulfide bonds and Cys has cationic and anionic groups.
This may be related to the large size and hydrophilicity of Cys,
which may weaken the hydrophobic adsorption of the enzyme on
the support.
3.7. Effect on enzyme specificity
3.5.2. Effect on enzyme stability
In many different papers it has been shown that the lipase prop-
erties may be easily tuned by altering the enzyme structure in a
small measure [41–47,54,55]; in some instances some prepara-
tions are almost inactive versus p-NPB while they are the most
Before selecting an optimal preparation, the effect of the
blocking step on enzyme stability was analyzed at pH
(Fig. 3)[22,23,27,30,39].
5
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active versus other more complex substrates (e.g., [56]) For this rea-
son, the activities of the different enzyme preparations have been
compared versus different substrates, which are structurally very
different.
The selected substrates have been ethyl hexanoate with an acyl
group having an aliphatic acid (both, in a biphasic system or in
the presence of solvent to have a homogenous solution), methyl
phenylacetate, with an aromatic ring, and R or S methyl mandelate,
a chiral molecule having an aromatic ring with a hydroxy group in
alpha position. Tables 2–4 show the results.
from 0.76 to 0.96, except if blocked with Cys that gave a value of
0.56.
The results above show that a blocking reagent for an enzyme
may be the one enabling the highest activity with one substrate,
while the situation may be very different if using another sub-
strate. This complex situation makes that these results are not easy
to explain or predict, the hypothesis used in many publications
is that the enzyme suffer some distortions or some limitation to
the movements that produce different enzyme conformations, and
therefore, different enzyme catalytic properties [22]. This means
that the determination of the best preparation for a specific reaction
needs to be determined by trial and error. However, even if these
is an empirical method that may sound inelegant, the results show
that the blocking may be an additional tool to strongly modulate
the enzyme specificity and that way to increase the library of avail-
able biocatalyst, increasing the possibilities of getting a suitable
biocatalysts for an specific process [47].
Using OCDVS-RML (Table 2), ethyl hexanoate in a biphasic sys-
tem (just aqueous buffer) was hydrolyzed in the most rapid fashion
when blocked with ETA and Gly, Asp gave almost as bad results as
the thiolated compounds, while EDA gave only 60% of the activity
compared to the best blocking reagents. In the presence of organic
solvents, all of them decreased the activity. The best results were
obtained using Asp, followed by ETA, Gly and also EDA. Asp activity
was even slightly increased in the presence of the organic solvent.
If methyl phenylacetate was the substrate, the most active prepa-
ration was that blocked with Gly, while the second one was that
blocked with Asp. Moreover, the preparation blocked with mer-
captoethanol was more active than the one blocked with EDA (in
opposition to the results using the aliphatic substrate). Using man-
delic esters, Gly blocked preparation is again the most active, but
now the second is the one blocked with Asp and the third with
ETA, while the less active preparation is that blocked with mercap-
toethanol.
Table 3 shows the results using CRL. Hydrolyzing ethyl hex-
anoate in biphasic medium, the most active preparation is that
blocked with Gly, almost doubling the second that is blocked with
ETA. Cys blocked preparation is the least active. Adding solvent
to have a monophasic phase, all systems decreased the activity
except that blocked with Asp, which became the most active shortly
followed by the preparation blocked with Gly. The activity with
phenylacetate is so low that we have not reliable values. Using the
esters of mandelic acid, the activity is low, but it is possible to deter-
mine it. The enzyme hydrolyzed the S isomer more rapidly than the
R isomer. Considering the S isomer, the most active is the prepara-
tion blocked with Gly, followed by that blocked with propanethiol
and after, the one blocked with Asp, ETA and Cys. Considering the R
isomer, the most active preparation is that blocked with Cys (it was
the worst with the aliphatic ester or p-NPB). This produced that the
ratio between reactions rates with both isomer go from 0.2 using
the preparation blocked with Gly to 0.44 using that blocked with
Cys.
4. Discussion
The new OCDVS supports have proved to be very suitable to
solve the problems that the OC preparation may have, e.g., unde-
sired enzyme desorption under certain conditions. Moreover, they
have widely improved the results obtained using OCGLX in terms of
activity recovery and stability. The possibility of almost full enzyme
desorption after the first immobilization at pH 5 confirmed that
the first immobilization of lipases on OCDVS supports was via
interfacial activation and not a covalent reaction via the vinylsul-
fone groups on the support. OCDVS is able to produce a covalent
bond between the already adsorbed enzyme and the vinylsulfone
groups on the support by incubation at pH 8, avoiding the risk
of enzyme desorption and the enzyme inactivation produced by
the incubation of RML and CRL at pH 10 (required when using
OCGLX)[18]. The increase in the incubation time of the OCDVS-
supports permits to increase the enzyme stability, suggesting that
some multipoint covalent attachment may be obtained and per-
mitting that the final preparations were much more stable than
the OCGLX (e.g., 20 folds in the case of CRL). However, it seems
that if the enzyme adsorbed on OC supports is distorted during
attachment, this has a negative effect on the further enzyme stabil-
ity (perhaps due to the near presence of the octyl layer), and that
is not compensated by the likely introduction of some additional
covalent bonds.
The blocking step has been found to be not only a compulsory
step to stop the enzyme-support reaction, but it has been revealed
as a potent tool to further tuning the enzyme features. There
are many reports showing that immobilization may alter lipase
properties[41–47,54,55], but there not so many reports showing
how the change of the final properties of the support surface may
greatly alter enzyme properties [23,40]. Here, we have shown that
stability and specificity of the 3 enzymes could be modified by
the blocking step, even considering that the support have mainly
octyl groups, and although in terms of stability the optimal block-
ing for each enzyme seem to be defined, in terms of activity it has
been found that the best blocking reagent strongly depends on the
substrate. The results above show that a blocking reagent for an
enzyme may be the one enabling the highest activity with one sub-
strate, while the situation may be very different if using another
substrate. This complex situation makes that these results are not
easy to explain or predict, the hypothesis used in many publica-
tions is that the enzyme suffer some distortions or some limitation
to the movements that produce different enzyme conformations,
and therefore, different enzyme catalytic properties, in fact these
different confirmation has been shown in some papers [22]. This
means that the determination of the best preparation for a spe-
OCDVS-TLL also is greatly modulated by the blocking step
(Table 4). Using the aliphatic ester, the preparation blocked with
mercaptoethanol, followed by those blocked with ETA and Gly
are the most active ones, the least active is that blocked with
propanethiol. In the presence of solvent, the biocatalyst blocked
with propanethiol is the one that decreases its activity to a lesser
degree and becomes the most active together with the prepara-
tion blocked with mercaptoethanol. In these conditions, the least
active preparation is that blocked with ETA. Using methyl phenyl
acetate, the most active preparation is that blocked with Gly,
followed by that blocked with ETA and EDA, the least active prepa-
ration is that blocked with Cys. These preparations also hydrolyzed
more rapidly the S isomer of methyl mandelate. The differences
in activity are very short for the R isomer (0.0299 U/mg for the
preparation blocked with Gly, 0.022 U/mg for that blocked with
mercaptoethanol), the only that is clearly less active is that blocked
with Cys. Using the S isomer, differences are similar (0.0356 for that
prepared using Gly, 0.0238 using EDA), again the blocking with Cys
produce the less active preparation. The ratio of activities versus
both isomer is also no very depended on the blocking, only move
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Table 2
Activity of different OCDVS- RML biocatalyst versus different substrates. The reactions were performed as described in Methods section.
Biocatalyst
VHexanoate
V Hexanoate(25%ACN)
V Methylphenylacetate
V R-methylmandelate
V S–methylmandelate
VR/VSmethylmandelate
(Biphasicphase)
OCDVS-RML-glycine
87.0000 0.609
90.0000 0.630
52.5000 0.368
5.0000 0.035
12.0000 0.084
19.5000 0.137
4.5000 0.032
12.6000 0.088
11.8500 0.083
10.6500 0.075
0.1475 0.001
12.9000 0.090
4.7025 0.033
0.5700 0.004
0.0026 0.000
0.0015 0.000
0.0012 0.000
0.0006 0.000
0.0019 0.000
0.0009 0.000
0.0013 0.000
0.0271 0.003
0.0248 0.002
0.0150 0.002
0.0058 0.001
0.0260 0.003
0.0049 0.000
0.0036 0.000
0.0281 0.002
0.0273 0.002
0.0202 0.001
0.0083 0.001
0.0317 0.002
0.0048 0.000
0.0039 0.000
0.96
0.91
0.74
0.70
0.82
1.02
0.90
OCDVS-RML-ethanolamine
OCDVS-RML-ethylenediamine
OCDVS-RML-cysteine
OCDVS-RML-aspartic acid
OCDVS-RML-propanethiol
OCDVS-RML-mercaptoethanol
Table 3
Activity of different OCDVS- CRL biocatalyst versus different substrates. Experimental details may be found in Methods section.
Biocatalyst
VHexanoate
V Hexanoate(25%ACN)
V Methylphenylacetate
V R-methylmandelate
V S-methylmandelate
VR/VSmethylmandelate
(Biphasicphase)
OCDVS-TLL-glycine
411.111 32.889
418.5185 33.481
351.8519 28.148
305.5556 24.444
361.1111 28.889
203.7037 16.296
425.9259 34.074
76.6667 3.833
38.3333 1.917
56.666 2.833
59.7778 2.989
65.4444 3.272
96.1111 4.806
96.4444 4.822
0.0298 0.003
0.0207 0.002
0.0192 0.002
0.0056 0.001
0.0182 0.002
0.0083 0.001
0.0106 0.001
0.0299 0.002
0.0273 0.002
0.0229 0.002
0.0093 0.001
0.0280 0.002
0.0243 0.002
0.0220 0.002
0.0356 0.002
0.0333 0.002
0.0238 0.002
0.0165 0.001
0.0367 0.003
0.0318 0.002
0.0260 0.002
0.84
0.82
0.96
0.56
0.76
0.76
0.84
OCDVS-TLL-ethanolamine
OCDVS-TLL-ethylenediamine
OCDVS-TLL-cysteine
OCDVS-TLL-aspartic acid
OCDVS-TLL-propanethiol
OCDVS-TLL-mercaptoethanol
Table 4
Activity of different OCDVS- TLL biocatalyst versus different substrates. Experimental details may be found in Methods section.
Biocatalyst
VHexanoate
V Hexanoate(25%ACN)
V Methylphenylacetate
V R-methylmandelate
V S-methylmandelate
VR/VSmethylmandelate
(Biphasicphase)
OCDVS-TLL-glycine
411.1111 32.889
418.5185 33.481
351.8519 28.148
305.5556 24.444
361.1111 28.889
203.7037 16.296
425.9259 34.074
76.6667 3.833
38.3333 1.917
56.6667 2.833
59.7778 2.989
65.4444 3.272
96.1111 4.806
96.4444 4.822
0.0298 0.003
0.0207 0.002
0.0192 0.002
0.0056 0.001
0.0182 0.002
0.0083 0.001
0.0106 0.001
0.0299 0.002
0.0273 0.002
0.0229 0.002
0.0093 0.001
0.0280 0.002
0.0243 0.002
0.0220 0.002
0.0356 0.002
0.0333 0.002
0.0238 0.002
0.0165 0.001
0.0367 0.003
0.0318 0.002
0.0260 0.002
0.84
0.82
0.96
0.56
0.76
0.76
0.84
OCDVS-TLL-ethanolamine
OCDVS-TLL-ethylenediamine
OCDVS-TLL-cysteine
OCDVS-TLL-aspartic acid
OCDVS-TLL-propanethiol
OCDVS-TLL-mercaptoethanol
cific reaction needs to be determined by trial and error. However,
even if these is an empirical method that may sound inelegant,
the results show that the blocking may be an additional tool to
strongly modulate the enzyme specificity and that way to increase
the library of available biocatalyst, increasing the possibilities of
getting a suitable biocatalysts for an specific process [47].
We want to point out that the very good results on the immo-
bilization of RML on OCDVS support are in contradiction with the
very bad results obtained in the immobilization of RML on DVS
activated supports [27] and show that heterofunctional supports
may be used to overcome some difficulties of standard supports.
Moreover, the stabilizing effects of the covalent attachment using
DVS activated Oc agarose are much higher that the stabilizing effect
of the immobilization of the enzymes on pure “DVS” activated sup-
ports, showing again the great interest of the combination of having
interfacially activated [22,27] lipases and a rigidification via cova-
lent attachment.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.procbio.2016.04.
002.
References
[1] K. Jaeger, Microbial lipases form versatile tools for biotechnology, Trends
Biotechnol. 16 (1998) 396–403.
[2] P. Villeneuve, J.M. Muderhwa, J. Graille, M.J. Haas, Customizing lipases for
biocatalysis: a survey of chemical, physical and molecular biological
approaches, J. Mol. Catal. B Enzym. 9 (2000) 113–148.
[3] A. Ghanem, Trends in lipase-catalyzed asymmetric access to enantiomerically
pure/enriched compounds, Tetrahedron 63 (2007) 1721–1754.
[4] P. Reis, K. Holmberg, H. Watzke, M.E. Leser, R. Miller, Lipases at interfaces: a
review, Adv. Colloid Interface Sci. 147-148 (2009) 237–250.
[5] C. Carvalho, Reverse micelles as reaction media for lipases, Biochimie 82
(2000) 1063–1085.
Thus, OCDVS seems to be a very suitable tool to improve and
tune the lipase properties.
[6] U. Hanefeld, L. Gardossi, E. Magner, Understanding enzyme immobilisation,
Chem. Soc. Rev. 38 (2009) 453–468.
[7] R.A. Sheldon, S. van Pelt, Enzyme immobilisation in biocatalysis: why, what
and how, Chem. Soc. Rev. 42 (2013) 6223–6235.
[8] P. Adlercreutz, Immobilisation and application of lipases in organic media,
Chem. Soc. Rev. 42 (2013) 6406–6436.
[9] R. DiCosimo, J. McAuliffe, A.J. Poulose, G. Bohlmann, Industrial use of
immobilized enzymes, Chem. Soc. Rev. 42 (2013) 6437–6474.
[10] A. Liese, L. Hilterhaus, Evaluation of immobilized enzymes for industrial
applications, Chem. Soc. Rev. 42 (2013) 6236–6249.
[11] M.B. Ansorge-Schumacher, O. Thum, Immobilised lipases in the cosmetics
industry, Chem. Soc. Rev. 42 (2013) 6475–6490.
[12] O. Barbosa, R. Torres, C. Ortiz, A. Berenguer-Murcia, R.C. Rodrigues, R.
Fernandez-Lafuente, Heterofunctional supports in enzyme immobilization:
from traditional immobilization protocols to opportunities in tuning enzyme
properties, Biomacromolecules 14 (2013) 2433–2462.
[13] C. Bernal, A. Illanes, L. Wilson, Heterofunctional hydrophilic-hydrophobic
porous silica as support for multipoint covalent immobilization of lipases:
application to lactulose palmitate synthesis, Langmuir 30 (2014) 3557–3566.
[14] C. Bernal, A. Illanes, L. Wilson, Improvement of efficiency in the enzymatic
synthesis of lactulose palmitate, J. Agric. Food Chem. 63 (2015) 3716–3724.
Acknowledgments
We gratefully recognize the support from MINECO, grant
CTQ2013-41507-R and COST Action CM1303 “Systems Biocataly-
sis”. The predoctoral fellowships for Mr de Albuquerque and Dr.
dos Santos (CNPq, Brazil) and Ms. Rueda (Colciencias, Colombia
and Becas Iberoamérica “Jóvenes Investigadores”, Banco San-
tander) are also recognized. The authors wish to thank Mr. Ramiro
Martínez (Novozymes, Spain) for kindly supplying the enzymes
used in this research. The help and comments from Dr. Ángel
Berenguer (Instituto de Materiales, Universidad de Alicante) are
kindly acknowledged.
Please cite this article in press as: T.L.de Albuquerque, et al., Easy stabilization of interfacially activated lipases using heterofunctional
divinyl sulfone activated-octyl agarose beads. Modulation of the immobilized enzymes by altering their nanoenvironment, Process
Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.04.002

G Model
PRBI-10653; No. of Pages10
ARTICLE IN PRESS
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[15] A. Suescun, N. Rueda, J.C.S. dos Santos, J.J. Castillo, C. Ortiz, R. Torres, et al.,
Immobilization of lipases on glyoxyl–octyl supports: improved stability and
reactivation strategies, Process Biochem. 50 (2015) 1211–1217.
[16] N. Guajardo, C. Bernal, L. Wilson, Z. Cabrera, Asymmetric hydrolysis of
dimethyl-3-phenylglutarate in sequential batch reactor operation catalyzed
by immobilized Geobacillus thermocatenulatus lipase, Catal. Today 255 (2015)
21–26.
[17] N. Guajardo, C. Bernal, L. Wilson, Z. Cabrera, Selectivity of R-␣-monobenzoate
glycerol synthesis catalyzed by Candida antarctica lipase B immobilized on
heterofunctional supports, Process Biochem. 50 (2015) 1870–1877.
[18] N. Rueda, J.C.S. dos Santos, R. Torres, C. Ortiz, O. Barbosa, R.
Fernandez-Lafuente, Improved performance of lipases immobilized on
heterofunctional octyl-glyoxyl agarose beads, RSC Adv. 5 (2015)
11212–11222.
[36] P. Prikryl, J. Lenfeld, D. Horak, M. Ticha, Z. Kucerova, Magnetic bead cellulose
as a suitable support for immobilization of ␣-chymotrypsin, Appl. Biochem.
Biotechnol. 168 (2012) 295–305.
[37] M.D. Bale Oenick, S.J. Danielson, J.L. Daiss, M.W. Sunderberg, R.C. Sutton,
Antigen-binding activity of antibodies immobilized on styrene copolymer
beads, Ann. Biol. Clin. (Paris) 48 (1990) 651–654.
[38] E. Ilan, P.G. Morton, T.M.S. Chang, The anaerobic reaction of bovine
hemoglobin with divinyl sulfone: structural changes and functional
consequences, Biochim: Biophys. Acta—Protein Struct. Mol. Enzymol. 1163
(1993) 257–265.
[39] J.C.S. dos Santos, N. Rueda, O. Barbosa, M.C. Millán-Linares, J. Pedroche, M.M.
Yuste, et al., Bovine trypsin immobilization on agarose activated with
divinylsulfone: improved activity and stability via multipoint covalent
attachment, J. Mol. Catal. B Enzym. 117 (2015) 38–44.
[19] E.A. Manoel, J.C.S. dos Santos, D.M.G. Freire, N. Rueda, R. Fernandez-Lafuente,
Immobilization of lipases on hydrophobic supports involves the open form of
the enzyme, Enzyme Microb. Technol. 71 (2015) 53–57.
[20] K.E. Jaeger, S. Ransac, H.B. Koch, F. Ferrato, B.W. Dijkstra, Topological
characterization and modeling of the 3D structure of lipase from Pseudomonas
aeruginosa, FEBS Lett. 332 (1993) 143–149.
[40] P. Bonomi, T. Bavaro, I. Serra, A. Tagliani, M. Terreni, D. Ubiali, Modulation of
the microenvironment surrounding the active site of penicillin g acylase
immobilized on acrylic carriers improves the enzymatic synthesis of
cephalosporins, Molecules 18 (2013) 14349–14365.
[41] A. Chaubey, R. Parshad, S. Koul, S.C. Taneja, G.N. Qazi, Enantioselectivity
modulation through immobilization of Arthrobacter sp. lipase: kinetic
resolution of fluoxetine intermediate, J. Mol. Catal. B Enzym. 42 (2006) 39–44.
[42] A. Chaubey, R. Parshad, P. Gupta, S.C. Taneja, G.N. Qazi, C.R. Rajan, et al.,
Arthrobacter sp lipase immobilization for preparation of enantiopure masked
beta-amino alcohols, Bioorg. Med. Chem. 17 (2009) 29–34.
[43] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R.
Fernandez-Lafuente, Improvement of enzyme activity, stability and
selectivity via immobilization techniques, Enzyme Microb. Technol. 40 (2007)
1451–1463.
[44] J. Liu, S. Bai, Q. Jin, H. Zhong, C. Li, Q. Yang, Improved catalytic performance of
lipase accommodated in the mesoporous silicas with polymer-modified
microenvironment, Langmuir 28 (2012) 9788–9796.
[45] J. Brem, M.C. Turcu, C. Paizs, K. Lundell, M.I. Toa, F.D. Irimie, et al.,
Immobilization to improve the properties of Pseudomonas fluorescens lipase
for the kinetic resolution of 3-aryl-3-hydroxy esters, Process Biochem. 47
(2012) 119–126.
[21] M. Cygler, J.D. Schrag, Structure and conformational flexibility of Candida
rugosa lipase, Biochim. Biophys. Acta 1441 (1999) 205–214.
[22] J.C.S. dos Santos, N. Rueda, A. Sanchez, R. Villalonga, L.R.B. Gonc¸ alves, R.
Fernandez-Lafuente, Versatility of divinylsulfone supports permits the tuning
of CALB properties during its immobilization, RSC Adv. 5 (2015) 35801–35810.
[23] J.C.S. dos Santos, N. Rueda, L.R.B. Gonc¸ alves, R. Fernandez-Lafuente, Tuning
the catalytic properties of lipases immobilized on divinylsulfone activated
agarose by altering its nanoenvironment, Enzyme Microb. Technol. 77 (2015)
1–7.
[24] R. Fernandez-Lafuente, P. Armisén, P. Sabuquillo, G. Fernández-Lorente, J.M.
Guisán, Immobilization of lipases by selective adsorption on hydrophobic
supports, Chem. Phys. Lipids 93 (1998) 185–197.
[25] C. Mateo, O. Abian, M. Bernedo, E. Cuenca, M. Fuentes, G. Fernandez-Lorente,
et al., Some special features of glyoxyl supports to immobilize proteins,
Enzyme Microb. Technol. 37 (2005) 456–462.
[26] N. Rueda, J.C.S. dos Santos, R. Torres, O. Barbosa, C. Ortiz, R.
Fernandez-Lafuente, Reactivation of lipases by the unfolding and refolding of
covalently immobilized biocatalysts, RSC Adv. 5 (2015) 55588–55594.
[27] J.C.S. dos Santos, N. Rueda, R. Torres, O. Barbosa, L.R.B. Gonc¸ alves, R.
Fernandez-Lafuente, Evaluation of divinylsulfone activated agarose to
immobilize lipases and to tune their catalytic properties, Process Biochem. 50
(2015) 918–927.
[28] N. Rueda, J.C.S. dos Santos, C. Ortiz, O. Barbosa, R. Fernandez-Lafuente, R.
Torres, Chemical amination of lipases improves their immobilization on
octyl-glyoxyl agarose beads, Catal. Today 259 (2016) 107–118.
[29] M. Galvis, O. Barbosa, R. Torres, C. Ortiz, R. Fernandez-Lafuente, Effect of
solid-phase chemical modification on the features of the lipase from
Thermomyces lanuginosus, Process Biochem. 47 (2012) 460–466.
[30] J.C.S. dos Santos, N. Rueda, O. Barbosa, J.F. Fernández-Sánchez, A.L.
Medina-Castillo, T. Ramón-Márquez, et al., Characterization of supports
activated with divinyl sulfone as a tool to immobilize and stabilize enzymes
via multipoint covalent attachment Application to chymotrypsin, RSC Adv. 5
(2015) 20639–20649.
[46] J.M. Palomo, Modulation of enzymes selectivity via immobilization, Curr. Org.
Synth. 6 (2009) 1–14.
[47] R.C. Rodrigues, C. Ortiz, Á. Berenguer-Murcia, R. Torres, R.
Fernández-Lafuente, Modifying enzyme activity and selectivity by
immobilization, Chem. Soc. Rev. 42 (2013) 6290–6307.
[48] P. Domínguez De María, J.M. Sánchez-Montero, J.V. Sinisterra, A.R. Alcántara,
Understanding Candida rugosa lipases: an overview, Biotechnol. Adv. 24
(2006) 180–196.
[49] R.C. Rodrigues, R. Fernandez-Lafuente, Lipase from Rhizomucor miehei as a
biocatalyst in fats and oils modification, J. Mol. Catal. B Enzym. 66 (2010)
15–32.
[50] R.C. Rodrigues, R. Fernandez-Lafuente, Lipase from Rhizomucor miehei as an
industrial biocatalyst in chemical process, J. Mol. Catal. B Enzym. 64 (2010)
1–22.
[51] R. Fernandez-Lafuente, Lipase from Thermomyces lanuginosus: Uses and
prospects as an industrial biocatalyst, J. Mol. Catal B Enzym. 62 (2010)
197–212.
[52] M. Bradford, Rapid and sensitive method for quantification of microgram
quantities of protein utilizing principle of protein-dye-binding, Anal.
Biochem. 72 (1976) 248–254.
[53] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head
of bacteriophage T4, Nature 227 (1970) 680–685.
[31] J. Porath, T. Laas, J.C. Janson, Agar derivatives for chromatography,
electrophoresis and gel-bound enzymes III. Rigid agarose gels cross-linked
with divinyl sulphone (dvs), J. Chromatogr. 103 (1975) 49–62.
[32] F.J. Lopez-Jaramillo, M. Ortega-Munõz, A. Megia-Fernandez, F.
Hernandez-Mateo, F. Santoyo-Gonzalez, Vinyl sulfone functionalization: a
feasible approach for the study of the lectin-carbohydrate interactions,
Bioconjug. Chem. 23 (2012) 846–855.
[54] A. Sugihara, M. Ueshima, Y. Shimada, S. Tsunasawa, Y. Tominaga, Purification
and characterization of a novel thermostable lipase from Pseudomonas
cepacia, J. Biochem. 112 (1992) 598–603.
[33] M. Ortega-Mun˜oz, J. Morales-Sanfrutos, A. Megia-Fernandez, F.J.
Lopez-Jaramillo, F. Hernandez-Mateo, F. Santoyo-Gonzalez, Vinyl sulfone
functionalized silica: a ready to use pre-activated material for immobilization
of biomolecules, J. Mater. Chem. 20 (2010) 7189–7196.
[34] A.L. Medina-Castillo, J. Morales-Sanfrutos, A. Megia-Fernandez, J.F.
Fernandez-Sanchez, F. Santoyo-Gonzalez, A. Fernandez-Gutierrez, Novel
synthetic route for covalent coupling of biomolecules on super-paramagnetic
hybrid nanoparticles, J. Polym. Sci. Part A Polym. Chem. 50 (2012) 3944–3953.
[35] J. Morales-Sanfrutos, J. Lopez-Jaramillo, M. Ortega-Mun˜oz, A.
Megia-Fernandez, F. Perez-Balderas, F. Hernandez-Mateo, et al., Vinyl sulfone:
a versatile function for simple bioconjugation and immobilization, Org.
Biomol. Chem. 8 (2010) 667–675.
[55] A. Tomin, D. Weiser, G. Hellner, Z. Bata, L. Corici, F. Péter, et al., Fine-tuning
the second generation sol-gel lipase immobilization with ternary alkoxysilane
precursor systems, Process Biochem. 46 (2011) 52–58.
[56] C. Garcia-Galan, O. Barbosa, K. Hernandez, J.C.S. dos Santos, R.C. Rodrigues, R.
Fernandez-Lafuente, Evaluation of styrene-divinylbenzene beads as a support
to immobilize lipases, Molecules 19 (2014) 7629–7645.
Please cite this article in press as: T.L.de Albuquerque, et al., Easy stabilization of interfacially activated lipases using heterofunctional
divinyl sulfone activated-octyl agarose beads. Modulation of the immobilized enzymes by altering their nanoenvironment, Process
Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.04.002