Reactivation of lipases by the unfolding and refolding of covalently immobilized biocatalysts

Article abstract of DOI:10.1039/c5ra07379k

Lipases from Candida antarctica (isoform B) (CALB) and Thermomyces lanuginosus (TLL) have been immobilized either covalently or by interfacial activation versus an octyl support, followed by covalent attachment by glyoxyl groups using octyl-glyoxyl agarose beads (OCGLX). These biocatalysts have been submitted to successive cycles of unfolding by incubation in 9 M guanidine and refolding by incubation in aqueous 100 mM phosphate buffer at pH 7, before and after total inactivation in the presence of organic solvents. The four preparations have been reactivated to some extent using this strategy, but the results depended on the method of preparation. Glyoxyl-immobilized CALB may recover 100% of its activity versus p-nitrophenyl butyrate, but after solvent inactivation the recovery of activity was reduced to 95%. The pure covalent TLL preparation recovered around 80% of its activity, either before or after solvent inactivation. Both enzymes showed less recovery of activity using OCGLX, as might be expected from the hydrophobic nature of the supporting groups (60% for CALB and 45% for TLL). In addition, using enzymes previously inactivated by solvent, recovery decreased by 5-10%. These values were maintained through three successive cycles. However, using R- and S-methyl mandelate, it was clear that recovery of activity decreased with further reactivation cycles. Taken as a whole, the unfolding and refolding effect may be used to recover a degree of enzyme activity. This is relevant in terms of application, as it may allow the enzyme preparations to be used for a longer period. However, to reach a similar enzyme structure in each reactivation cycle, it will be necessary to undertake further studies involving the use of other supports in order to improve the unfolding and refolding steps.

Full text of DOI:10.1039/c5ra07379k

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DOS SANTOS, R. Torres, O. Barbosa, C. ORTIZ and R. FERNANDEZ LAFUENTE, RSC Adv., 2015, DOI:
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REFOLDED
IMMOBILIZED
PROTEIN??
OCGLX—lipase:
REACTIVATION STRATEGIES
AQUEOUS MEDIUM
OCGLX
IMMOBILIZED
PROTEIN
9 M
GUANIDINE
ORGANIC SOLVENT
DISTORTED
IMMOBILIZED
PROTEIN??
UNFOLDED IMMOBILIZED
PROTEIN
Glyoxyl
Octyl

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DOI: 10.1039/C5RA07379K
REACTIVATION OF LIPASES VIA UNFOLDING/REFOLDING OF COVALENTLY
IMMOBILIZED BIOCATALYSTS.
Nazzoly Ruedaa,b,+, Jose C. S. dos Santosa,c.+, Rodrigo Torresb,d, Oveimar Barbosae,
Claudia Ortizf,*, and Roberto FernandezꢀLafuentea,*.
a: ICPꢀCSIC. Campus UAMꢀCSIC. Cantoblanco. 28049 Madrid. Spain.
b: Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Edificio
Camilo Torres 210, Universidad Industrial de Santander, CEP 680001 ,Bucaramanga, Colombia.
c: Departamento de Engenharia Química, Universidade Federal Do Ceará, Campus Do Pici, CEP
60455ꢀ760, Fortaleza, CE, Brazil.
d: Current address: Laboratorio de Biotecnología, Instituto Colombiano del PetróleoꢀEcopetrol,
Piedecuesta, Bucaramanga, Colombia.
e: Grupo de investigación en productos naturales (GIPRONUT) Departamento de Química,
Facultad de Ciencias. Universidad del Tolima, Ibagué, Colombia
f: Escuela de Microbiología, Universidad Industrial de Santander, Bucaramanga, Colombia
+ Both authors have evenly contributed to this paper
*Coꢀcorresponding authors:
Claudia Ortiz
Roberto FernandezꢀLafuente
GIBIM
Departamento de Biocatálisis.
Instituto de CatálisisꢀCSIC
Escuela de Microbiología
UIS
C/ Marie Curie 2.
Bucaramanga
Campus UAMꢀCSIC. Cantoblanco
28049 Madrid (Spain).
rfl@icp.csic.es
Colombia
Eꢀmail: ortizc@uis.edu.co

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Abstract
Lipases from Candida antarctica (isoform B) (CALB) and Thermomyces lanuginosus (TLL)
have been immobilized covalently or via interfacial activation versus octyl support followed by
covalent attachment via glyoxyl groups using octylꢀglyoxyl agarose beads (OCGLX). These
biocatalysts have been submitted to successive cycles of unfolding by incubation in 9 M guanidine
and refolding by incubation in aqueous 100 mM phosphate buffer at pH 7, before and after total
inactivation in the presence of organic solvents. The four preparations may be reactivated in some
extension using this strategy, but results depended on the preparation. Glyoxyl immobilized CALB
may recover 100% of activity versus the pꢀnitrophenyl butyrate, but after solvent inactivation this
activity recovery was only 95%. The pure covalent TLL preparation permitted to recover around
80% of activity after or before solvent inactivation. Both enzymes offer lower activity recoveries
using OCGLX, as may be expected from the hydrophobic nature of the groups in the support (60%
for CALB and 45% for TLL). Moreover, using previously solvent inactivated enzymes, the results
decreased in a 5ꢀ10%. These values were maintained along three successive cycles. However, using
R and S methyl mandelate, it is clear that the activity recovery decreased along the reactivation
cycles. Altogether, the unfolding/refolding strategy may be used to obtain part of the enzyme
activity and that is relevant from an applied point of view, as this may permit to use the enzyme
preparations for longer times. However, to reach the same enzyme structure in each reactivation
cycle it is necessary to perform further studies that may involve from use of other supports to
improve the unfolding and refolding steps of this strategy.
Key words: Enzyme reactivation, operation stability, enzyme solvent inactivation, enzyme
unfolding, enzyme refolding, octylꢀglyoxyl supports, heterofunctional support.

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Introduction
Enzymes are very interesting biocatalysts due to their high activity in aqueous media at low
temperature and pressure, and high enantio and regio selectivity and specificity 1–7
.
However, enzymes have also some limitations, because the physiological optimal properties
and the features required for their industrial implementation do not match in some points8. This
occurs for example with the moderate stability of enzymes at conditions far from the physiological
9,10
ones, that in many times are the ones required by industry
. The operational enzyme stability
may be improved by genetic tools 11, chemical modifications (e.g., crosslinking of the enzyme
surface) 12,13, enzyme immobilization (e.g., generating adequate nanoꢀenvironments or via
multipoint or multisubunit immobilization)14–18, and also by selecting adequate reaction conditions
19
.
Enzyme immobilization is a requirement for many industrial applications 20, and it is
compatible with any other strategy of enzyme stabilization13,18,21,22. Moreover, the biocatalyst
operational stability may be further improved if the inactivated biocatalyst may be submitted to
strategies of reactivation after partial or total inactivation23. If the enzyme is incubated in the
presence of inert organic solvents, at neutral pH value and moderate temperature, the enzyme will
be inactivated mainly via the promotion of incorrect structures. In these cases, the enzyme may be
submitted to strategies of unfolding/refolding, and the native structure of the enzyme may be
recovered 24
.
Some recent papers suggest that the previous immobilization of the enzymes on supports via
covalent linkages may help the refolding step25–28. Together with the simplification promoted by
the use of immobilized enzymes, the immobilization prevent the enzyme aggregation during any
step of the unfolding/refolding process, and if several enzymeꢀsupport linkages are established, the
refolding may be facilitated because the relative positions of these groups cannot be altered, and

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they may act as reference points during refolding 29. The main requirements to use this strategy are
that the enzyme molecules remain attached to the support during the whole protocol, and that the
support was inert enough to avoid undesired enzymeꢀsupport interactions. Surprisingly, even fully
chemically aminated enzymes could be refolded after immobilization via multipoint covalent
attachment 29,30
.
Some of the examples of reactivation of immobilized enzymes involve lipases 26,29–31. These
enzymes have a peculiar mechanism of action, the so called interfacial activation, which requires
some movements of the enzyme structure between a closed structure, with a polypeptide chain
(called lid) blocking the active center, and an open structure, with the active center exposed to the
medium32–36. This lipase open form is the only active one in many cases, and has a tendency to
become adsorbed on hydrophobic surfaces37. The lid may have different configurations, in the case
of the popular lipase from Candida antarctica (form B) (CALB) the lid is very small and does not
fully isolate the active center from the medium 38, but CALB is still able to become adsorbed on
39
hydrophobic surfaces
. In other cases, like the lipase from Bacillus thermocatenulatus, the
40
enzyme has a double lid . However, usually the lipases have a large single lid able to fully
prevent the interaction of enzyme active center and medium, like in the case of the lipase from
Thermomyces lanuginosus (TLL) 41
.
On the other hand, one very useful strategy to immobilize lipases is the interfacial activation
of the enzyme on octyl agarose 42,43. This strategy has permitted the one step immobilization /
purification / hyperactivation / stabilization of many lipases 44. However, this immobilization
protocol is not compatible with unfolding/refolding reactivation strategies, as this immobilization is
reversible 44. In fact, the incubation of the lipase immobilized on this support in solutions having
high concentration of guanidine is a strategy used to recover the support after enzyme inactivation
45
44. However, recently a new heterofunctional support has been proposed, octylꢀglyoxyl agarose
.
This support couples the advantages of octyl agarose to those of the covalent attachment, making
the immobilization irreversible. Moreover, these new biocatalysts are usually even more stable than

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the standard octylꢀlipase biocatalysts 45. As a drawback, it should be considered that using this
support the immobilization becomes irreversible, and therefore the support cannot be reused after
enzyme inactivation. However, now it may be possible to submit the immobilized enzyme to
unfolding/refolding reactivation strategies without risk of enzyme desorption, making it
unnecessary to discard neither enzyme nor support 45. A likely problem is the possibility of
interaction of the hydrophobic groups of the enzyme with the octyl groups of the support during
refolding that could avoid the full reactivation of the enzyme.
In this new paper, the reactivation possibilities of CALB and TLL covalently immobilized
on standard supports (glyoxyl o cyanogen bromide) or immobilized on octyl glyoxyl supports have
been studied and compared. CALB is the most utilized lipase in literature, with many
48
applications46,47, and presents a very small lid38, while TLL is also very utilized in literature , and
has a large lid41.

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2. Materials and methods
2.1. Materials
Solutions of CALB (6.9 mg of protein /mL) and TLL (36 mg of protein /mL) were a kind gift
from Novozymes (Spain). Cyanogen bromide crosslinked 4 % agarose (CNBr) beads and octylꢀ
agarose beads were from GE Healthcare. R and S methyl mandelate, pꢀnitrophenyl butyrate (pꢀ
NPB) were from Sigma Chemical Co. (St. Louis, MO, USA). All reagents and solvents were of
analytical grade. The preparation of octylꢀglyoxyl and glyoxylꢀagarose was performed as previously
described 45,49.
2.2. Standard determination of enzyme activity
This assay was performed by measuring the increase in absorbance at 348 nm produced by
the released pꢀnitrophenol in the hydrolysis of 0.4 mM pꢀNPB in 100 mM sodium phosphate at pH
7.0 and 25 °C (ɛ under these conditions is 5150 M−1 cm−1). To start the reaction, 50–100 ꢁL of
lipase solution or suspension were added to 2.5 mL of substrate solution. One international unit of
activity (U) was defined as the amount of enzyme that hydrolyzes 1 ꢁmol of pꢀNPB per minute
under the conditions described previously. Protein concentration was determined using Bradford’s
method 28 and bovine serum albumin was used as the reference.
2.3. Immobilization of enzymes
2.3.1 Immobilization of enzymes on octyl-glyoxyl (OCGLX) supports
The immobilization was performed using 1 or 5 mg of protein per g of wet support. The
commercial samples of the enzymes were diluted in the corresponding volume of 5 mM sodium

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45
phosphate at pH 7. Then, the OCGLX support was added. The activity of both supernatant and
suspension was followed using pꢀNPB. After immobilization the suspension was filtered and the
supported enzyme was washed several times with distilled water. Then, the washed immobilized
enzyme was reꢀsuspended at pH 10 for 12 h, to favor the enzymeꢀsupport covalent reaction 50.
2.3.2 Immobilization of enzymes on glyoxyl (GLX) support
The immobilization was performed using 1 or 5 mg of protein per g of wet support. The
enzymes were diluted in 50 mM sodium bicarbonate buffer at pH 10. Then, the support was
suspended in the enzyme solution under gentle stirring. Periodically, samples of the supernatant and
suspension were withdrawn, and the enzyme activity was measured as described above.
2.3.3 Reduction with sodium borohydride
To end the enzymeꢀsupport covalent reaction, solid sodium borohydride was added to a
concentration of 1 mg/mL to the OCGLX and GLX suspensions (at pH 10) and were submitted to
gentle stirring for 30 min. This treatment reduces reversible Schiff´s bases to very stable secondary
amino bonds and unreacted aldehydes groups to fully inert hydroxy groups49–51. The preparations
were washed with CTAB (TLL) or Triton (CALB) to eliminate the nonꢀcovalently attached enzyme
molecules. Finally the biocatalysts were filtered, washed with abundant distilled water and stored at
4°C.
2.3.4 Immobilization of enzymes on (BrCN) support
TLL could not be immobilized on glyoxyl agarose because the enzyme was not stable
29,48
enough at pH 10,
for this reason an alternative support was utilized. A volume of 2 mL of
commercial TLL was diluted in 60 mL of 5 mM sodium phosphate at pH 7. Then, 6 g of wet BrCNꢀ

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support was added. After 90 min at 4°C under gentle stirring, around 45% of lipase became
immobilized on the support. The enzyme–support immobilization was ended by incubating the
biocatalyst with 1 M ethanolamine at pH 8 for 2 h. Finally, the immobilized preparation was
washed with abundant distilled water and stored at 4°C.
2.4. Inactivation of different enzyme preparations in the presence of organic co-solvents
Enzyme preparations were incubated in mixtures of 90% (v/v) dimethylformamide (DMF)
(for CALB) or 80% (v/v) 1,4ꢀdioxane (for TLL) with 100 mM Tris–HCl pH 7 at 30°C.
Periodically, samples were withdrawn and the activity was measured using pꢀNPB as described
above. The organic coꢀsolvents presented in the samples did not have a significant effect during
enzyme activity determination (results not shown).
2.5. Incubation in sodium guanidine
Immobilized CALB and TLL biocatalysts were incubated in 9 M guanidine at 25°C for 12
hours. Then, the biocatalysts were filtered and washed with 100 mM sodium phosphate buffer at pH
7.0 to remove the denaturant, and resuspended in the same volume of aqueous 100 mM sodium
phosphate buffer at pH 7.0. Activity was periodically determined for 24 h.
2.6. Determination of the hydrolysis of R and S methyl mandelate
Enzyme activity was also determined using R or S methyl mandelate. A mass of 200 mg of
the immobilized preparations were added to 1 mL of 50 mM substrate in 50 mM sodium phosphate
at pH 7 and 25 °C under continuous stirring. The conversion degree was analyzed by RPꢀHPLC
(Spectra Physic SP 100 coupled with an UV detector Spectra Physic SP 8450) using a Kromasil

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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 ammonium acetate (35:65, v/v) at pH 2.8 as mobile phase and
UV detection was performed at 230 nm. The acid presented a retention time of 2.4 minutes while
the ester had a retention time of 4.2 minutes. One unit of enzyme activity was defined as the amount
of enzyme necessary to produce 1 ꢁmol of mandelic acid per minute under the conditions described
above. Activity was determined by triplicate with a maximum conversion of 20–30%, and data are
given as average values.

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3. Results
3.1. Unfolding/Refolding of immobilized TLL and CALB
Figure 1 shows that GLXꢀCALB may be submitted to 3 consecutive cycles of
unfolding/refolding steps recovering 100% of the initial activity versus pꢀNPB. The reactivation
was very rapid and in one hour the enzyme fully recovered the initial activity. Using OCGLXꢀ
CALB the situation is not so positive. The reactivation permitted to recover 60% of the initial
activity in the first cycle, and the reactivation is slower than using GLX. In the third cycle, the
reactivation gave a 55% of the initial enzyme activity.
The results point that GLXꢀCALB, that by the nature of immobilization on GLX supports
involves a multipoint attachment50 and provides a fully inert surface, permits a rapid and almost
complete refolding of the enzyme: the multipoint covalent attachment provides some reference
points to properly refold the enzyme 29 while the inert surface avoids any undesired enzymeꢀsupport
interaction that could stabilize different enzyme structures. Using OCGLX, the reactivation is
slower and did not reach the initial activity (60%). This may be mainly caused for some interactions
between the unfolded enzyme structures during the enzyme refolding and the octyl groups of the
support. Even if we cannot fully recover the enzyme activity, OCGLXꢀCALB recovered a large
percentage of activity in a consistent way, and that is another advantage of this new preparation
when compared to just octyl supports, where all enzyme molecules are released from the support if
incubated under unfolding conditions (results not shown).
Figure 2 shows the results using TLL. This enzyme cannot be immobilized on GLX because
its low stability at pH 1029,48, therefore as a standard covalent reference for our studies, BrCNꢀ
agarose has been used. This preparation permitted the recovery of around 82% of the enzyme
activity, and the reactivation took several hours. In the third cycle, the reactivation enables the
recovering of 78% of the enzyme activity, very similar to the values of the first cycle. Using
OCGLXꢀTLL, again a lower percentage of initial enzyme activity is recovered, the 3 cycles ranged

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between 48% and 45%. Moreover, reactivation is slower than using BrCNꢀTLL. These results
suggested that the refolding of TLL may be more complex than that of CALB, and agree with
previous reports, where the addition of some detergents improved the reactivation results 31.
3.2. Reactivation of immobilized TLL and CALB
Next, we have studied the possibilities of this strategy to get the reactivation of enzyme that
have been inactivated by the action of organic solvents. If we are able to fully unfold the enzyme
structure, the results should be similar to that obtained after unfolding/refolding of the unaltered
immobilized enzyme.
Figure 3 shows that reactivation of GLXꢀCALB after full inactivation in the presence of
90% DMF (v/v) permitted to recover 95% of the enzyme activity in the first cycle and 90% in the
third one. Moreover, the reactivation was slower than that shown in Figure 1. Results were similar
using OCGLXꢀCALB; now reactivation accounted for only 50% in the first cycle and 45% in the
third one, versus the 60% observed using the unaltered enzyme. In any case, the OCGLX
immobilized enzyme operational live could be increased, and this may revert in part the lack of
reversibility of the immobilization using this support.
Reactivation of BrCNꢀTLL permitted to recover similar values of enzyme activity after
enzyme inactivation in the presence of 80% dioxane (v/v) (Figure 4) to those obtained when the
unaltered enzyme was used (Figure 2), around 80% of enzyme activity was recovered during 3
successive inactivation/reactivation cycles. However, using OCGLXꢀTLL results now (Figure 4)
differed from those in Figure 2: reactivation permitted to recover only around 40% of the initial
activity, although this value was maintained along the 3 studied cycles.
The results were not identical comparing the unaltered immobilized enzyme and the
inactivated enzymes, except for BrCNꢀTLL. Using GLXꢀCALB, one likely explanation is that we

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cannot fully unfold this multipoint immobilized enzyme even in 9 M guanidine, and therefore the
structures that the enzyme has when refolding starts are not identical and drive to different final
structures. The presence of large octyl groups and the attachment of the enzyme via very short
spacer arms may produce some additional problems to the unfolding and refolding steps of the
reactivation strategy. A likely explanation for the good results using BrCNꢀTLL is that this
preparation has very few enzymeꢀsupport attachments (very likely just one) and the unfolding and
refolding is not hampered by the interactions with the support.
3.3. Activity of different treated CALB and TLL immobilized enzymes versus R and S
methyl mandelate.
The proposed strategy of unfolding/refolding of enzymes covalently immobilized has
proved to be quite useful, but leaves some doubts on the real form of the reactivated enzyme
structure and if this form may be obtained during several reactivation cycles. pꢀNPB is a very easily
hydrolyzed ester, and it has been the only substrate used in many of the published papers. Now, we
have evaluated the immobilized enzymes after the different treatments in the hydrolysis of a more
complex substrate, both isomers of the chiral methyl mandelate. Results are collected in Table 1. It
is clear that the percentages of activity recovered using these more complex substrates are lower
than those obtained using pꢀNPB, and also decreased when comparing cycles 1 and cycles 3 of the
unfolding/refolding, suggesting that the structure of the enzyme that we obtain in each reactivation
cycle may differ that obtained in the previous cycle. Thus, OCGLXꢀCALB decrease the activity
versus Rꢀmethyl mandelate to less than 70% in the first cycle, and after 3 cycles 60% of the activity
were recovered. The inactivation of the enzyme by incubation in 90% DMF did not significantly
alter the results. Using GLXꢀCALB, in opposition with the results using pNPB, now a first decrease
in activity to 80% is clear. After the third cycle, only 70% of the initial activity is reovered.
OCGLXꢀTLL decrease the activity to 50% in the first cycle of unfolding/refolding experiments and

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to a similar value if the enzyme was first inactivated with dioxane, after 3 cycles the recovered
activity decreased slight in both cases. BrCNꢀTLL preparations presented a very similar behavior.
Using CALB, the decrease of enzyme activity usually is higher versus the isomer that is the
worst substrate (the S isomer) and this fact produced an increase of the CALB enantiospecificity in
this reaction after applying the reactivation strategy. OCGLXꢀCALB increased the ratios of the rate
of hydrolysis of isomers R and S from 2 to a maximum of 2.7. It may be expected that the not fully
recovering of the enzyme structure has a higher impact in the activity versus the less suitable
substrate.
This result agrees with the discrepancy between the results obtained in the
unfolding/refolding of unaltered and inactivated enzyme preparations and points that even if this
simple strategy may be useful to reactivate enzymes used in simple reactions with good substrates,
it may be more complex if the enzymes are used with worse substrates, as the achievement of an
identically functional enzyme structure seems unfulfilled.
4.-Discussion
The strategy of unfolding/refolding may be used to reactivate inactivated enzymes by the
incubation in high concentrations of organic solvents if they are covalently attached to the support.
This is possible even using a support whose surface is full of large and hydrophobic octyl groups,
which produces diverse problems to the unfolding/refolding of the enzyme, offering lower
percentages of activity recovery. However, enzyme activity and stability in these supports is very
adequate and this possibility for partial reactivation may further increase the usefulness of the
immobilization method compared to the use of pure octyl supports (where reactivation is not
possible) or covalent preparations (where activity and stability is lower).

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However, the results point out that it is not simple to reach the initial enzyme structure, or at
least an identical structure after each reactivation cycle, and that may be a problem if the enzyme is
used in reactions where the enzyme determines not only the reaction rates, but also the quality of
the product (e.g, in resolution of racemic mixtures). Moreover, this suggests that in studies of
reactivation of enzymes, it is not enough to use just a simple and easily modifiable substrate, some
substrates hardly recognized by the enzymes may be used a probes of the real reactivation of the
enzyme molecules.
The lack of a full recovery of the enzyme activity or at least a similar activity during the
successive cycles suggests that we are not obtaining exactly the same structure on each refolding
cycle. Apparently, simpler substrates permit a higher enzyme conformation change before the
activity start to decrease. The failure in recovering similar activity in each reactivation cycle may
have several explanations First, an enzyme submitted to unfolding/refolding strategies may give a
not fully renatured enzyme, even in free form, and that may produce an enzyme with different
conformation and, therefore, different functional properties52. Moreover, Illanes´s group has
reported how reactivated immobilized penicillin G acylase biocatalysts really have a significant
alteration of the kinetic parameters compared to the initial enzyme preparations, although they have
not analyzed if this new enzyme structure was always obtained during successive reactivation
cycles, as just one cycle was studied53.
It has been also reported that if unfolding/refolding strategies are applied to immobilized
enzymes, if the distance between enzyme molecules is not large enough to prevent interactions
between unfolded enzyme structures (unfolded state have a larger volume that folded one), these
interactions may prevent a correct refolding of the unfolded enzyme25. This problem cannot occur
in the covalent preparation, where the immobilization is very slow and the loading of the support
was quite low, but immobilization of lipases on OCGLX is so rapid that can form a crown in the
outer part of the support particle of enzyme molecules packed together. In fact, it has been shown

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that even using very low enzyme loading, the distance between enzyme molecules immobilized on
octyl supports is so short that may be crosslinked using a molecule as small as glutaraldehyde54,55
.
Thus, it is very likely that OCGLX preparations have diverse problems for the refolding:
interactions with the octyl groups, some steric problems of the movements of the protein chain
generated by the support and a close proximity of other unfolded enzyme molecules than can
produce undesired interactions. However, it is clear also that the enzyme activity may be fully
destroyed by enzyme distortion caused by incubation in solvents, unfolded by incubation in
guanidine, and we can get a high percentage of enzyme activity after refolding in aqueous medium.
Thus, these initial promising results, showing a reactivation of lipases immobilized on
OCGLX supports open new lines of research, trying to shortcut some of the problems found in this
research to apply unfolding/refolding strategies as a method of reactivating lipases immobilized in
this interesting support. Optimization of the reactivation medium (e.g., using detergents, thiolated
compounds) may permit to further improve these promising results.
Acknowledgments
We gratefully recognize the support from the MINECO of Spanish Government, CTQ2013ꢀ
41507ꢀR. The predoctoral fellowships for Ms. Rueda (Colciencias, Colombian Goberment) and Mr
dos Santos (CNPq, Brazil) 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.

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Figure legends
Figure 1. Cycles of unfolding -refolding of different biocatalyst from CALB. Unfolding has
been carried out via incubation in 9M guanidine and refolding by incubation in aqueous 100 mM
sodium phosphate . Experiments have been performed as described in Section 2. Circles: OCGLX,
Squares: GLX.
Figure 2. Cycles of unfolding -refolding of different biocatalyst from TLL. Unfolding has been
carried out via incubation in 9M guanidine and refolding by incubation in aqueous medium.
Experiments have been performed as described in Section 2. Circles: OCGLX, Squares: BrCN.
Figure 3. Cycles of enzyme inactivation by incubation in 90% DMF and reactivation by
unfolding via incubation in 9 M guanidine and refolding by incubation in aqueous medium of
different biocatalyst from CALB. Experiments have been performed as described in Section 2.
Circles: OCGLX, Squares: GLX.
Figure 4. Cycles of enzyme inactivation by incubation in 80% dioxane and reactivation by
unfolding via incubation in 9 M guanidine and refolding by incubation in aqueous medium of
different biocatalyst from TLL. Experiments have been performed as described in Section 2.
Circles: OCGLX, Squares: BrCN.

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DOI: 10.1039/C5RA07379K
BIOCATALYST
OCGLXCALB
OCGLXCALB* Cycle 1
OCGLXCALB *Cycle 3
OCGLXCALB ** Cycle 1
OCGLXCALB ** Cycle 3
VR-methyl mandelate
VS-methyl mandelate
VR/VS
83 ± 4.2
57 ± 2.9
51.2 ± 2.7
53 ± 2.7
42 ± 2.1
21.1 ± 1.1
19.7 ± 1
23 ± 1.2
20 ± 1
2.0
2.7
2.6
2.3
2.5
49.3 ± 2.5
GLXCALB
23.7 ± 1.2
19.1 ± 1
17.2 ± 0.9
17.1 ± 0.9
16.3 ± 0.7
11.3 ± 0.6
7.6 ± 0.4
7.1 ± 0.4
7.6 ± 0.4
7.2 ± 0.4
2.1
2.5
2.4
2.3
2.3
GLXCALB* Cycle 1
GLXCALB* Cycle 3
GLXCALB ** Cycle 1
GLXCALB** Cycle 1
OCGLXTLL
0.034 ± 0.002
0.017 ± 0.001
0.015 ± 0.001
0.019 ± 0.002
0.018 ± 0.002
0.031 ± 0.002
0.011 ± 0.001
0.009 ± 0.001
0.012 ± 0.001
0.010 ± 0.001
1.1
1.5
1.7
1.6
1.8
OCGLXTLL* Cycle 1
OCGLXTLL* Cycle 3
OCGLXTLL** Cycle 1
OCGLXTLL** Cycle 3
BrCNTLL
0.0088 ± 0.0002 0.0058 ± 0.0002
0.0047 ± 0.0001 0.0031 ± 0.0001
0.0046 ± 0.0001 0.0031 ± 0.0001
0.0045 ± 0.0001 0.0032 ± 0.0001
0.0041 ± 0.0001 0.0028 ± 0.0001
1.5
1.5
1.5
1.4
1.5
BrCNTLL* Cycle 1
BrCNTLL* Cycle 3
BrCNTLL ** Cycle 1
BrCNTLL ** Cycle 3
Table 1. Activity of different biocatalysts versus R or S methyl mandelate (50 mM) at pH 7
and 25°C. Experiments were performed as described in section 2. The activity (V) is given in
ꢁmoles of substrate hydrolyzed per minute and mg of immobilized enzyme.
* The biocatalyst has been submitted to unfolding/refolding for the indicated number of
cycles.
** The biocatalyst has been submitted to inactivation by incubation in solvent (90% DMF
for CALB, 80% dioxane for TLL), unfolding and refolding steps during the indicated
number of cycles.