Stabilizing effects of cations on lipases depend on the immobilization protocol

Article abstract of DOI:10.1039/c5ra18344h

The effect of an additive on enzyme stability used to be considered an intrinsic feature of a lipase. However, in this paper we have found that the effect of additive on enzyme stability depends on the immobilization protocol. After assaying the effects of diverse chloride salts with different cations on different lipases activity, no relevant effect was detected. Free enzymes or the covalently immobilized enzymes are not stabilized by these cations for any of the studied lipases. However, Mn²⁺ and Ca²⁺ (at a concentration of 5 mM) are able to greatly stabilize the lipases from Rhizomucor miehei (RML) and Candida rugosa (CRL) when they are present during the inactivation, but only if the enzymes are immobilized on octyl-agarose (stabilization factor ranging from 20 to 50). The effect was only detected when using more than 2.5 mM of the cations, and reached the maximum value at 5 mM, suggesting a saturation mechanism of action. The stabilization seemed to be based on a specific mechanism, and required the recognition sites to be saturated by the cations. Mg²⁺ has no effect on enzyme stability for both enzymes, but it is able to suppress the stabilization promoted by the other two cations using CRL; while it has no effect on the cation stabilization when using RML. This is the first report of a cation induced enzyme stabilization effect that depends on the lipase immobilization protocol.

Full text of DOI:10.1039/c5ra18344h

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LAFUENTE, RSC Adv., 2015, DOI: 10.1039/C5RA18344H.
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STABILIZING EFFECTS OF CATIONS ON LIPASES DEPEND ON THE
IMMOBILIZATION PROTOCOL
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a,+
a,+
a,b
Laura FernandezꢀLopez , Rocio BartolomeꢀCabrero , Maria Daniela Rodriguez , Cleiton S.
a,c
a,d;*
a
Dos Santos , Nazzoly Rueda
and Roberto FernandezꢀLafuente *
a
Departamento de Biocatálisis. Instituto de CatálisisꢀCSIC, Campus UAMꢀCSIC Madrid. Spain.
Laboratorio de Biotecnología Molecular. Instituto de Biotecnología Misiones "María Ebe
b
Reca". (FCEQyN – UnaM). Posadas, Argentina.
c
Departamento de Engenharia Química, Universidade Federal Do Ceará, Campus Do Pici, CEP
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60455ꢀ760, Fortaleza, CE, Brazil.
d
Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM),
Edificio Camilo Torres 210, Universidad Industrial de Santander, Bucaramanga, Colombia.
+:Both authors have contriobuted evenly to this paper.
*Coꢀcorresponding authors:
Nazzoly Rueda
Roberto FernandezꢀLafuente
Departamento de Biocatálisis.
Instituto de CatálisisꢀCSIC
C/ Marie Curie 2.
Escuela de Química
Universidad Industrial de Santander
Bucaramanga, Santander
COLOMBIA
Campus UAMꢀCSIC. Cantoblanco.
28049 Madrid (Spain).
nazzoly@gmail.com
rfl@icp.csic.es
1

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Abstract
The effect of an additive on enzyme stability use to be considered an intrinsic feature of
the lipase. However, in this paper we have found that the effect of additive on enzyme stability
is depended on the immobilization protocol. After assaying the effects of diverse chloride salts
with different cations on different lipases activity, no relevant effect was detected. Free enzymes
or the covalently immobilized enzymes are not stabilized by these cations for any of the studied
2
+
2+
lipases. However, Mn and Ca (at a concentration of 5 mM) are able to greatly stabilize the
lipases from Rhizomucor miehei (RML) and Candida rugosa (CRL) when they are present
during the inactivation, but only if the enzymes are immobilized on octylꢀagarose (stabilization
factor ranging from 20 to 50). The effect was only detected using more than 2.5 mM of the
cations, and reached the maximum value at 5 mM, suggesting a saturation mechanism of action.
The stabilization seemed to be based on a specific mechanism, and required to have the
2
+
recognition sites saturated by the cations. Mg has no effect on enzyme stability for both
enzymes, but it is able to suppress the stabilization promoted by the other two cations using
CRL; while it has no effect on the cation stabilization when using RML.
This is the first report of a cation induced enzyme stabilization effect that depends on the
lipase immobilization protocol.
Key words: Lipase stabilization, octylꢀagarose, interfacial activation, additives, cations.
2

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1.- Introduction
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Lipases are very promising catalysts, with the capability to catalyze a broad range of
1
–7
reactions ranging from ester hydrolysis to amide synthesis , lipases are also able to catalyze
some promiscuous reactions unrelated to their biological activity and even to their active center
8
–12
. Lipases are robust in different reaction media, do not require any cofactor and admit many
1
–7
different substrates while exhibiting a high enantio or regio specificity and selectivity
.
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The improvement of enzyme stability is always a way to enhance the handling of the any
enzyme, lipases included. An enhanced lipase stability also increases the range of conditions
1
3,14
where the lipase may be used and the operational lifetime of the biocatalyst
. Lipase stability
improvement may be achieved via genetic tools (siteꢀdirected mutagenesis or directed evolution)
1
2
5–18
9
19–25
26–
, chemical modification or immobilization
, or the use of some stabilizing additives
. The use of additives presents some economical and process development drawbacks if used
during the final industrial reaction catalyzed by the lipase; they need to be added during each
reaction cycle and may complicate the final reaction product purification. These problems are
lower during lipase handling, and in fact most industrial soluble enzyme preparations are
30–32
stabilized by mixtures of different compounds, e,g., saccharides of different nature
.
Immobilization of enzymes is performed in many instances in the presence of inhibitors or other
enzyme stabilizers to avoid the loss of activity associated in some instances to the different steps
3
3–35
of an immobilization protocol
.
Cations may be very simple and cheap additives. The effect of cations on enzyme
36–
activity is systematically analyzed in the characterization of new enzymes in many instances
3
9
. However, the effect of cations on enzyme stability, mainly if the actions lack of effect on
enzyme activity, is not usually considered, although in some cases the cation effects are quite
40–43
relevant
. Ions may be very relevant to stabilize the enzyme surface ionic net, forming some
ionic crosslinking on specific areas or stabilizing concrete areas by ionic bridges. There are
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many reports on enzymes that are stabilized by some specific cations, mainly multimeric
44,45
proteins where the cation plays a critical role in stabilizing the multimeric structure
, and
other cases where a stabilization is achieved by the use of a high ionic strength, independently of
the ions used. Nevertheless, it may be assumed that if a low concentration (e.g., 5 mM) of an ion
has a great positive effect on enzyme stability, this may be used under any circumstance without
great problem.
Moreover, the stabilization effect of adequate cations may be coupled to the
stabilization achieved via other strategies, like immobilization. In fact, in some instances the
role of an ion on enzyme stability could only be disclosed when some inactivation causes were
46
eliminated via immobilization (e.g., preventing multimeric enzyme subunit dissociation) .
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On the other hand, lipases exist in two different forms. The closed form has the active
center isolated from the environment by a polypeptide chain, called lid or flat. The lid has a very
hydrophobic internal face that interacts with the hydrophobic areas surrounding the active
center. In the open and active form of lipases, the lid moves and exposes the active center to the
medium. This open form tends to become adsorbed on hydrophobic surfaces and become
47–50
stabilized (the soꢀcalled interfacial activation)
. Thus, it is likely that the effect of the cations
may differ depending on the use of a lipase interfacially activated versus a hydrophobic surface
or lipase formulations where the conformational equilibrium exists. Free enzyme always has the
risk of precipitation in the presence of multivalent cations, where lipases also have a tendency to
51,52
form bimolecular aggregates
formulations.
and conclusions may be difficult to reach with these enzyme
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In this paper, the effect of different cations on the stability of 3 different preparations
5
3,54
of two of the most popular lipases, those from Rhizomucor miehei (RML)
and from
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Candida rugosa (CRL)
have been studied.
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The effects of the cations on the free enzymes, the enzymes covalently immobilized,
where the conformational equilibrium is maintained, and on octylꢀagarose, where the open form
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of the lipases becomes stabilized have been analyzed. The main objective of the study is too
analyze if the effect of an additive is an inherent property of an enzyme, or may be greatly
modulated if the immobilization protocol produce different enzyme conformations, as in the
current research effort.
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2.- MATERIALS AND METHODS
2.1. Materials
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Solutions of lipases from Candida antarctica (isoform A) (9.5mg of protein /mL)
(CALA), Thermomyces lanuginosus (TLL) (36 mg of protein /mL), Lecitase (16 mg of protein
/mL) and RML (13.7 mg of protein /mL) were a kind gift from Novozymes (Spain). Cyanogen
bromide crosslinked 4 % agarose (BrCN) beads and octylꢀagarose beads were from GE
Healthcare. pꢀNitrophenyl butyrate (pꢀNPB), diethyl pꢀnitrophenylphosphate (DꢀpNPP) and
CRL (in powder form, 40% of protein content) were from Sigma Chemical Co. (St. Louis, MO,
USA). All reagents and solvents were of analytical grade. All experiments were performed by
triplicate and the results are given as an average with the standard deviation.
2.2. 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 25 mM sodium phosphate
−1
−1
at pH 7.0 and 25 °C (ɛ under these conditions is 5150 M cm ). 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. Only to assess the effect of the
cations on enzyme activity, different salts (5 mM) were added to check the effect on the enzyme
58
activity. Protein concentration was determined using Bradford’s method and bovine serum
albumin was used as the reference.
2.3. Immobilization of enzymes
The enzymes were immobilized under conventional conditions; the cations used in this
study were not added in this step.
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2.3.1 Immobilization of enzymes on BrCN support
5 g of BrCN agarose beads was added to 50 mL of an enzyme solution (5 mg of
protein/g of wet support) in 25 mM sodium phosphate at pH 7 containing 0.1% (v/v) Triton Xꢀ
52,59
100 to have the monomeric form of the lipases
. After 90 min at 4°C under gentle stirring,
the biocatalysts were washed with distilled water and incubated in a solution of 1 M
ethanolamine at pH 8 for 2 h to block the remaining reactive groups in the support. Finally, the
immobilized preparations were washed with abundant distilled water and stored at 4°C. The
biocatalysts thus prepared have an immobilization yield of 50% maintaining almost intact the
51
activity (as has been previously described in other examples using this support).
2.3.2 Immobilization of enzymes on octyl (OC) supports
The immobilization was performed using 1 mg of protein per g of wet support. The
commercial samples of the enzymes were diluted or dissolved in the corresponding volume of 5
mM sodium phosphate at pH 7. Then, the OC support was added (10 g of support in 100 ml of
enzyme solution). 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 and stored at 4ºC. The enzyme activity increased after immobilization
53,57, 60ꢀ64
except in the case of CAL, as has been previously described
2.4. Thermal inactivation of different enzyme preparations
This experiment was performed to determine the effect of the cations on the enzyme
stability. Free enzyme or 1 g of immobilized lipases were suspended in 10 mL of 5 mM of
sodium acetate at pH 5 or Tris buffer at pH 7 at different temperatures, containing different
concentrations of different salts. Periodically, samples were withdrawn and the activity was
measured using pꢀNPB. Halfꢀlives were calculated from the observed inactivation courses.
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2.5. Hydrolysis of R and S methyl mandelate
This experiment was performed to determine the effect of the cations on the enzyme
activity and specificity versus complex substrates of chiral nature. 50 mM solutions of R or S
2 2
methyl mandelate in 100 mM glycine at pH 7 containing or not 5 mM of CaCl or MnCl were
prepared. Reaction was initialized by adding 1 g of the lipase preparations in 5 mL of the
substrate solution. The suspension was maintained at 25°C under continuous stirring. The
reaction was followed by RPꢀHPLC (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 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
retention times of the acid was 3 min and of the ester was 7 min. The enzyme activity is given in
ꢁmol of mandelic acid per minute under the conditions described above. Activity was
determined by triplicate, using a maximum conversion of 20–30%.The data are given as average
values
2.6. Irreversible inactivation of immobilized lipases by D-pNPP
Different lipaseꢀimmobilized preparations (1g) were suspended in 10 mL of different
o
concentrations of sodium phosphate buffer (from 10 mM to 1 M) solution at pH 7 and 25 C.
Then, DꢀpNPP was added up to a concentration of 1 mM. Samples of this suspension were
withdrawn periodically, and their activities were checked using the pꢀNPB assay.
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3.-Results
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3.1.- Effect of some cations on the stability of different lipase preparations.
Free and different immobilized preparations of some enzymes were inactivated at
several T and pH 7 in the presence of diverse chloride salts. OC preparations of TLL, CALB
and Lecitase kept their stabilities almost unaltered in the presence of the whole set of assayed
+
+
2+
2+
2+
cations (Na , K , Mg , Mn , Ca ) (Fig 1 S).
Using RML and CRL results are quite surprising. While both free enzymes were not
stabilized by any of the cations (in fact the inactivation was slightly more rapid in presence of
the cations using RML and slight slower using CRL) , the stabilization effect is very relevant
2
+
2+
using the octyl preparations when 5 mM of Mn or Ca were present in the inactivation
suspensions (see Figures 1 and 2). OCꢀRML stabilization by both cations was really impressive,
when the enzyme inactivated in the absence of these cations were almost inactive, the cations
permitted the octyl preparations to retain over 90% of the activity (see Figure 1). The
stabilization observed with both cations were similar (OCꢀRML halfꢀlive improved around 50
2
+
2+
folds using Mn and 40 folds using Ca . OCꢀCRL stabilization was slightly lower (25 folds
2
+
2+
using Ca and 30 folds using Mn ) (Figure 2). In any case, this stabilization permits a
qualitative jump: The enzyme in the presence of cations retained over 80% of the initial activity
while the reference had less than 20%.
To check if the lack of effect of the cations on the free enzyme was due to the
aggregation of the RML and CRL molecules in the presence of these bivalent cations, both
enzymes were covalently immobilized on BrCN. After immobilization, aggregation was
impossible, the mild covalent attachment still permitted the open/closed equilibrium, and the
effects of the cations should be directly reflected on enzyme stability. Using BrCNꢀRML
(Figure 1) or BrCNꢀCRL (Figure 2), both covalently immobilized enzyme preparations did not
become stabilized by any of these cations, results were even closer to the reference than using
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the free enzyme. The inhibition of the free enzymes by DꢀpNPP was also very similar in
presence or absence of these salts, suggesting that the percentage of lipase openꢀclosed
molecules remained unaltered.
The lack of effect of the cations on the stability of the free enzyme could be associated
to a promotion of enzyme aggregation, which could mask the stabilization effect. However, this
may not be the explanation using the covalently immobilized enzyme, where aggregation is no
longer possible. Thus, results suggested that the open forms of RML and CRL stabilized versus
2+
2+
the hydrophobic surface of octyl supports are the forms that are stabilized by Mn or Ca ,
while the nonꢀabsorbed lipases are not stabilized by them, even if the opening/closing equilibria
2
+
2+
is possible. Stabilization using Mn is slightly higher than using Ca for both enzymes. The
2
+
2+
2+
lack of effect of Mg suggested that the lipase stabilization produced by Mn and Ca may
follow a specific mechanism, perhaps involving adsorption of these cations on some specific
places of the enzyme surface.
To analyze if these stabilization effects could be extrapolated to other conditions, the
effect of these cations on the whole set of preparations of RML and CRL were assayed at pH 5.
Figures 3 and 4 show that the results were similar to those found at pH 7. Free RML inactivation
rate was accelerated in a clearer way at pH 5 than at pH 7 by the presence of all the cations
(Figure 3), while free CRL stability remained almost unaltered (Figure 4). BrCNꢀRML seemed
2+
to be marginally stabilized by Mn (a factor of lower than 2) (Figure 3), while BrCNꢀCRL
stability was not influenced by the studied cations (Figure 4). Again, a very clear stabilization
2+
2+
was observed using Mn and Ca for the OC preparations. Using OCꢀRML, stabilization was
2
+
2+
lower than that detected at pH 7 (20 folds using Ca and 25 folds using Mn ), while using
2
+
2+
CRL stabilization seem to be very similar (28 times using Ca and 32 folds using Mn . At
2
+
2+
alkaline pH values, both Ca and Mn presented a much reduced solubility; therefore we have
focused the study only in neutral and acid pH values.
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The use of 5 mM of these cations during the activity assay did not promote any
relevant effect on the lipase activity on any of the different preparations and lipases assayed
(Table 1S). Thus, the main effect of the cations was just the improved stability of the octyl
preparation.
We further tried to characterize this stabilization effect.
2
+
2+
3.2.- Effect of the concentration of Mn and Ca on the stabilization effect
The concentrations of both cations were varied from 0 to 25 mM at pH 7. For both
enzymes, the behavior was fairly similar with some differences. Figure 5 shows that at
concentrations of both cations of 1 mM or below, the stabilization effect were very small, at 2.5
2+
mM the stabilization was 10ꢀ15 folds (except using Ca and OCꢀRML), the increase of the
cation concentration to 5 mM permitted to greatly increase this stabilization effect while a
further increase of the cations concentrations to 25 mM produced a very slight increase in the
2
+
stabilizing effect. The highest increase in stability when going from 5 to 25 mM was using Mn
and OCꢀRML. In this case, the increase in stabilization factor was from 50 to 60. This result
suggests that the cation must saturate the stabilization place/places of the enzyme; otherwise the
stabilizing effect is not relevant. Moreover, after saturation of these sites, a further increase on
the cation concentration has no effect on the enzyme stability.
Next, we study the effect of the combined use of equimolecular mixtures of both
stabilizing cations. Using 5 mM of both cations, the results were very similar to the independent
use of each of the cations, for both enzymes (results not shown). These results suggest that the
effect of both cations were via the same mechanism and not additive. Using 2.5 mM of both
cations, where the theoretical site is not fully saturated with any of the cations, observed
stabilities were only marginally better that using the individual cations at 2.5 mM, and lower
2
+
2+
than that obtained using 5 mM. Thus, 5 mM of Mn or Ca seemed to be necessary to take full
advantage of the stabilization produced by these cations.
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+
2+
2+
3.3.- Effect of the concentration of Mg on the stabilizing effects of Mn and Ca
2+
2+
Figure 6 shows the stabilizing effect of 5 mM of Mn and Ca in the presence of
2
+
growing concentrations of Mg , the divalent cation that did not produce any stabilizing effect.
2
+
Using OCꢀCRL, the use of 5 mM de Mg is able to fully suppress the stabilizing effect of both
cations at both pH values. However, the results are very different using OCꢀRML, where the
2
+
stability is almost unaffected in the presence of Mg in the presence of 5 mM of the stabilizing
2
+
cations. This means that Mg may compete for the site(s) where the stabilizing cations are
adsorbed using OCꢀCRL, suggesting that it may become adsorbed on the same place or in
2
+
2+
another place that make the adsorption of Mn and Ca to the right place no longer possible
(by inducing some conformational change or blocking the entry of the other cations to the right
pocket). This hypothetic site of CRL will have an affinity higher than that of the enzyme for the
stabilizing cations. This cation did not modify the effect of the stabilizing cations using OCꢀ
2
+
2+
2+
RML. Thus, or the Mg cannot access to the RML sites where Mn
and Ca
have an
stabilizing role, or the affinity of these places of the enzyme by these cations is far higher than
2
+
that by Mg (perhaps by this reason we have not found a stabilizing effect of this).
3.5.- Effect of the presence of cations in the specificity of RML and CRL.
It has been reported in many instances that the lipase specificity may be easily altered
by any strategy that may alter the final conformation of the enzyme, for example the
6
0–62
immobilization via different protocols
. In the first section of this paper, we have showed
that the cations have not effect on the catalytic activity of the lipases in a very simple reaction,
the hydrolysis of pNPB. Now, the enzyme activity versus a more complex substrate is analyzed:
R or S methyl mandelate has been hydrolyzed using octylꢀRML or CRL in presence or absence
of CaCl
2 2
or MnCl . This compound has an aromatic ring and a hydroxyl group in the alpha
position. Table 1 shows that neither RML nor CRL are greatly affected by these stabilizing
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cations in their catalytic activities versus this substrates. This suggested that the stabilizing role
of these cations did not involve a conformational change of the octyl preparations, but just they
reinforce the overall enzyme structure.
4.- Conclusion
The immobilization protocol has been shown to have a critical role in the lipase
features. The same chemical modification or physical coating of differently immobilized
biocatalysts of lipases has been described to promote very different effects on the enzyme
5
1ꢀ54, 60–62
properties, like selectivity or specificity
. However, this is the first report where a
simple additive, like a cation, is found to produce a high stabilization of only some immobilized
preparations of an enzyme and this effect is not found or is much less intense on other enzyme
preparations, including the free enzyme. This may be caused by the very different forms of the
two immobilized enzyme preparations: the OCꢀlipase has the open form adsorbed and stabilized
57
on the hydrophobic surface of OC supports , while BrCN immobilized lipase maintains the
equilibrium between closed and open conformation (nonꢀstabilized by adsorption to a
hydrophobic surface). Thus, while studying the free enzyme, the researcher will deduce a lack of
2+
2+
stabilizing effect of Mn or Ca , correlated to a lack of effect on the enzyme activity and
specificity, using OCꢀimmobilized enzymes the stabilization may be measured in dozens folds,
constituting a real qualitative stabilization. This becomes a new advantage of the immobilization
5
7
of these two enzymes on octyl supports .
2+
2+
Thus, the use of 5 mM of salts containing Mn or Ca may be used as a way to
greatly increase the enzyme stability in aqueous media during biocatalyst operation at pH values
ranging from 5 to 7; alkaline pH could not be analyzed due to problems in the salts solubility.
This low cation concentration may have a low economic impact used even during operation, and
in some cases may not be a great problem for the downstream of the final product. Moreover,
this low concentration of these cations may be used during storage and handling of the OC
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immobilized RML or CRL, enlarging the range of conditions where the enzymes may be
utilized 63,64. This way, these results may open new possibilities for the use and handling of these
interesting immobilized enzymes. The mechanism of stabilization by cations of these OCꢀlipase
preparations require further studies to determine the real nature, perhaps using differential
scanning fluorimetry or by isothermal titration calorimetry.
From the results presented in this paper, it is more relevant that the researcher cannot
expect that a stabilizing (or perhaps a destabilizing) effect attributed to a particular compound is
an inherent feature of an enzyme in all their formulations. This makes necessary to perform the
study of stabilization/destabilization of an enzyme biocatalyst each time that the immobilization
protocol is changed. The results of this paper show that the extrapolation of one result from one
enzyme preparation to other preparation may be very risky.
Acknowledgements
We gratefully recognize the support from the MINECO from Spanish Government,
(project number CTQ2013ꢀ41507ꢀR). The predoctoral fellowships for Ms. Rueda (Colciencias,
Colombian Government and Becas Iberoamérica “Jóvenes Investigadores”, Banco Santander)
and for Ms Rodríguez (CONICET and SPU, Argentine Government) and Dr dos Santos (CNPq,
Brazil) are also recognized. The authors wish to thank Mr. Ramiro Martínez (Novozymes,
Spain) for kindly supplying some of 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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27
References:
3
3
3
3
28
29
30
31
1.
2.
3.
4.
5.
K. E. Jaeger and T. Eggert, Curr. Opin. Biotechnol., 2002, 13, 390–397.
R. Sharma, Y. Chisti and U. C. Banerjee, Biotechnol. Adv., 2001, 19, 627–662.
K. E. Jaeger and M. T. Reetz, Trends Biotechnol., 1998, 16, 396–403.
R. D. Schmid and R. Verger, Angew. Chemie Int. Ed., 1998, 37, 1608–1633.
332
33
A. Pandey, S. Benjamin, C. R. Soccol, P. Nigam, N. Krieger and V. T. Soccol,
Biotechnol. Appl. Biochem., 1999, 29 ( Pt 2), 119–131.
3
3
3
34
35
6.
J. Zhang, H. Shi, D. Wu, Z. Xing, A. Zhang, Y. Yang and Q. Li, Process Biochem., 2014,
49, 797–806.
336
337
338
339
340
341
7.
8.
9.
P. Adlercreutz, Chem. Soc. Rev., 2013, 42, 6406–36.
K. Hult and P. Berglund, Trends Biotechnol., 2007, 25, 231–238.
M. Svedendahl, K. Hult and P. Berglund, J. Am. Chem. Soc., 2005, 127, 17988–17989.
10. M. Kapoor and M. N. Gupta, Process Biochem., 2012, 47, 555–569.
11. C. Li, X.ꢀW. Feng, N. Wang, Y.ꢀJ. Zhou and X.ꢀQ. Yu, Green Chem., 2008, 10, 616.
12. E. Busto, V. GotorꢀFernández and V. Gotor, Chem. Soc. Rev., 2010, 39, 4504–4523.
3
3
42
43
13. K. M. Polizzi, A. S. Bommarius, J. M. Broering and J. F. ChaparroꢀRiggers, Curr. Opin.
Chem. Biol., 2007, 11, 220–225.
3
3
44
45
14. D. C. Demirjian, F. MorísꢀVaras and C. S. Cassidy, Curr. Opin. Chem. Biol., 2001, 5,
144–151.
3
46
47
15. F. H. Arnold and A. A. Volkov, Curr. Opin. Chem. Biol., 1999, 3, 54–59.
16. U. T. Bornscheuer and M. Pohl, Curr. Opin. Chem. Biol., 2001, 5, 137–143.
3
3
3
48
49
17. V. G. H. Eijsink, S. GÅseidnes, T. V. Borchert and B. Van Den Burg, Biomol. Eng.,
2005, 22, 21–30.
350
351
352
353
354
18. O. Kuchner and F. H. Arnold, Trends Biotechnol., 1997, 15, 523–530.
19. P. V. Iyer and L. Ananthanarayan, Process Biochem., 2008, 43, 1019–1032.
20. D. Brady and J. Jordaan, Biotechnol. Lett., 2009, 31, 1639–1650.
21. C. Ó’Fágáin, Enzyme Microb. Technol., 2003, 33, 137–149.
22. L. Gianfreda and M. R. Scarfi, Mol. Cell. Biochem., 1991, 100, 97–128.
1
5

RSC Advances
Page 16 of 28
View Article Online
DOI: 10.1039/C5RA18344H
3
55
56
23. S. S. Wong and L. J. C. Wong, Enzyme Microb. Technol., 1992, 14, 866–874.
3
24. S. Janecek, Process Biochem., 1993, 28, 435–445.
̆
3
3
57
58
25. V. Stepankova, S. Bidmanova, T. Koudelakova, Z. Prokop, R. Chaloupkova and J.
Damborsky, ACS Catal., 2013, 3, 2823–2836.
359
360
361
26. H. Zhao, J. Mol. Catal. B Enzym., 2005, 37, 16–25.
27. A. Pollard and R. G. Wyn Jones, Planta, 1979, 144, 291–298.
28. J. M. Obón, A. Manjón and J. L. Iborra, Enzyme Microb. Technol., 1996, 19, 352–360.
3
3
62
63
29. W. A. Jensen, J. M. Armstrong, J. De Giorgio and M. T. W. Hearn, Biochim. Biophys.
Acta ꢀ Protein Struct. Mol. Enzymol., 1996, 1296, 23–34.
3
3
64
65
30. S. de Cordt, M. Hendrickx, G. Maesmans and P. Tobback, Biotechnol. Bioeng., 1994, 43,
107–114.
3
3
66
67
31. S. De Cordt, I. Avila, M. Hendrickx and P. Tobback, Biotechnol. Bioeng., 1994, 44, 859–
865.
3
68
69
32. K. Gekko and S. Koga, J. Biochem., 1983, 94, 199–205.
3
33. R. M. Blanco and J. M. Guisan, Enzyme Microb. Technol., 1988, 10, 227–232.
3
3
70
71
34. C. M. Rosell, R. FernandezꢀLafuente and J. M. Guisan, Biocatal. Biotransformation,
1995, 12, 67–76.
3
3
72
73
35. G. Alvaro, R. FernandezꢀLafuente, R. Blanco and J. Guisan, Enzyme Microb. Technol.,
1991, 13, 210–214.
374
375
376
377
36. L. G. Foe and J. L. Trujillo, Arch. Biochem. Biophys., 1980, 199, 1–6.
37. J. C. Warren and S. G. Cheatum, Biochemistry, 1966, 5, 1702–7.
38. G. Žoldák, M. Sprinzl and E. Sedlák, Eur. J. Biochem., 2004, 271, 48–57.
39. P. H. Von Hippel and T. Schleich, Acc. Chem. Res., 1969, 2, 257–265.
3
3
78
79
40. D. B. Wright, D. D. Banks, J. R. Lohman, J. L. Hilsenbeck and L. M. Gloss, J. Mol. Biol.,
2002, 323, 327–344.
380
381
382
383
41. P. H. Von Hippel and K. Y. Wong, J. Biol. Chem., 1965, 240, 3909–3923.
42. K. D. Collins, Proc. Natl. Acad. Sci. U. S. A., 1995, 92, 5553–5557.
43. J. A. Harmony, P. J. Shaffer and R. H. Himes, J. Biol. Chem., 1974, 249, 394–401.
44. R. FernandezꢀLafuente, Enzyme Microb. Technol., 2009, 45, 405–418.
1
6

Page 17 of 28
RSC Advances
View Article Online
DOI: 10.1039/C5RA18344H
3
3
84
85
45. C. GarciaꢀGalan, O. Barbosa and R. FernandezꢀLafuente, Enzyme Microb. Technol.,
2013, 52, 211–7.
3
3
86
87
46. S. Kaddour, F. LópezꢀGallego, T. Sadoun, R. FernandezꢀLafuente and J. M. Guisan, J.
Mol. Catal. B Enzym., 2008, 55, 142–145.
3
88
47. R. Verger, Trends Biotechnol., 1997, 15, 32–38.
3
3
89
90
48. C. Cambillau, S. Longhi, A. Nicolas and C. Martinez, Curr. Opin. Struct. Biol., 1996, 6,
449–455.
391
392
393
49. A. M. Brzozowski, U. Derewenda, Z. S. Derewenda, G. G. Dodson, D. M. Lawson, J. P.
Turkenburg, F. Bjorkling, B. HugeꢀJensen, S. A. Patkar and L. Thim, Nature, 1991, 351,
491–494.
3
3
94
95
50. K. K. Kim, H. K. Song, D. H. Shin, K. Y. Hwang and S. W. Suh, Structure, 1997, 5, 173–
185.
3
3
96
97
51. G. FernándezꢀLorente, J. M. Palomo, M. Fuentes, C. Mateo, J. M. Guisán and R.
FernándezꢀLafuente, Biotechnol. Bioeng., 2003, 82, 232–7.
3
3
98
99
52. J. M. Palomo, C. Ortiz, G. FernándezꢀLorente, M. Fuentes, J. M. Guisán and R.
FernándezꢀLafuente, Enzyme Microb. Technol., 2005, 36, 447–454.
400
401
402
53. R. C. Rodrigues and R. FernandezꢀLafuente, J. Mol. Catal. B Enzym., 2010, 64, 1–22.
54. R. C. Rodrigues and R. FernandezꢀLafuente, J. Mol. Catal. B Enzym., 2010, 66, 15–32.
55. M. Cygler and J. D. Schrag, Biochim. Biophys. Acta, 1999, 1441, 205–14.
4
4
03
04
56. P. Domínguez De María, J. M. SánchezꢀMontero, J. V. Sinisterra and A. R. Alcántara,
Biotechnol. Adv., 2006, 24, 180–196.
4
4
05
06
57. E. A. Manoel, J. C. S. dos Santos, D. M. G. Freire, N. Rueda and R. FernandezꢀLafuente,
Enzyme Microb. Technol., 2015, 71, 53–57.
4
07
58. M. M. Bradford, Anal. Biochem., 1976, 72, 248–254.
4
4
08
09
59. J. M. Palomo, M. Fuentes, G. FernándezꢀLorente, C. Mateo, J. M. Guisan and R.
FernándezꢀLafuente, Biomacromolecules, 2003, 4, 1–6.
4
4
10
11
60. J. C. S. dos Santos, C. GarciaꢀGalan, R. C. Rodrigues, H. B. de Sant’Ana, L. R. B.
Gonçalves and R. FernandezꢀLafuente, Enzyme Microb. Technol., 2014, 60, 1–8.
412
413
414
61. C. GarciaꢀGalan, J. C. S. dos Santos, O. Barbosa, R. Torres, E. B. Pereira, V. C.
Corberan, L. R. B. Gonçalves, and R. FernandezꢀLafuente, Process Biochem., 2014, 49,
604–616.
4
4
15
16
62. O. Barbosa, M. Ruiz, C. Ortiz, M. Fernández, R. Torres and R. FernandezꢀLafuente,
Process Biochem., 2012, 47, 867–876.
1
7

RSC Advances
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4
4
17
18
63. A. Suescun, N. Rueda, J. C. S. dos Santos, J. J. Castillo, C. Ortiz, R. Torres, O. Barbosa
and R. FernandezꢀLafuente, Process Biochem., 2015, 50, 1211–1217.
4
4
19
20
64. N. Rueda, J. C. S. dos Santos, R. Torres, O. Barbosa, C. Ortiz and R. Fernandezꢀ
Lafuente, RSC Adv., 2015, 5, 55588–55594.
4
21
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Figure legends
Figure 1. Stability of different RML preparations in the presence of 5mM of different
chloride salts prepared in 5mM Tris HCl at pH 7. Panel A: soluble RML, Panel B: OCꢀ
RML, Panel C: BrCNꢀRML. The enzyme preparations were incubated at the indicated
temperatures: Soluble RML at 45°C, OCꢀRML at 45°C and BrCNꢀRML at 50°C. Other
specifications are described in section 2. Without addition of extraꢀcations (Pointed line),
Sodium (Stars), Potassium (Rhombus), Calcium (Triangles), Magnesium (Squares), Manganese
(Circles).
Figure 2. Stability of different CRL preparations in the presence of 5mM of different
chloride salts prepared in 5mM Tris HCl at pH 7. Panel A: soluble CRL, Panel B: OCꢀCRL,
Panel C: BrCNꢀCRL. The enzyme preparations were incubated at the indicated temperatures:
Soluble CRL at 50°C, OCꢀCRL at 60°C and BrCNꢀCRL at 55°C. Other specifications are
described in section 2. Without addition of extraꢀcation (Pointed line), Sodium (Stars),
Potassium (Rhombus), Calcium (Triangles), Magnesium (Squares), Manganese (Circles).
Figure 3. Stability of different RML preparations in the presence of 5mM of different
chloride salts prepared in 5 mM sodium acetate at pH 5. Panel A: soluble RML, Panel B:
OCꢀRML, Panel C: BrCNꢀRML. The enzyme preparations were incubated at the indicated:
Soluble RML at 45°C, OCꢀRML at 50°C and BrCNꢀRML at 65°C. Other specifications are
described in section 2. Without addition of extraꢀcation (Pointed line), Sodium (Stars),
Potassium (Rhombus), Calcium (Triangles), Magnesium (Squares), Manganese (Circles).
Figure 4. Stability of different CRL preparations in the presence of 5mM of different
chloride salts prepared in 5 mM sodium acetate at pH 5. Panel A: soluble CRL, Panel B:
OCꢀCRL, Panel C: BrCNꢀCRL. The enzyme preparations were incubated at indicated
temperatures: Soluble CRL at 50°C, OCꢀCRL at 58°C and BrCNꢀCRL at 60°C. Other
specifications are described in section 2. Without addition of extraꢀcation (Pointed line), Sodium
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448
449
450
451
452
453
454
455
456
457
458
459
460
(Stars), Potassium (Rhombus), Calcium (Triangles), Magnesium (Squares), Manganese
(Circles).
Figure 5. Effect of MnCl2 and CaCl , concentrations on the stability of OC-RML (Pointed
2
line) and OC-CRL (Solid black line). The unit of Relative Units is considered the half live of
the OC preparation in Tris without cations. The inactivation was performed in Tris HCL 5 mM
at pH 7 at the mentioned temperatures: RML assays were performed at 45°C and CRL assays at
60°C. Other specifications are described in section 2. CaCl (Triangles), MnCl (Circles).
2
2
Figure 6. Effect of the concentration of MgCl on the stabilizing effects of MnCl and
2
2
2
CaCl on OC-RML (Pointed line) and OC-CRL (Solid black line). The unit of Relative
Units is considered the stability of the OC preparation in Tris without cations. Enzyme
preparations were incubated at specified temperatures: RML assays were performed at 45°C and
CRL at 60°C. Other specifications are described in section 2. CaCl (Triangles), MnCl
2
2
(Circles).
2
0

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463
Table 1. Effect of diferent cations on the octyl-lipase activity versus R or S methyl
mandelate. Enzyme reactions were carried out using as substrate R or S methyl mandelate (50
mM) at pH 7 and 25 °C as described in Section 2.
*
*
Biocatalyst(cation) V R Methyl mandelate
V S Methyl mandelate
V / V S
R
OC RML (none)
6.5 ± 0.32
6.8 ± 0.34
7.2 ± 0.36
7.0 ± 0.35
6.1 ± 0.31
7.1 ± 0.36
0.93
1.11
1.01
OC RML (CaCl
2
)
OC RML (MnCl
2
)
OC CRL (none)
0.5 ± 0.03
0.6 ± 0.03
0.6 ± 0.03
1.4 ± 0.07
1.3 ± 0.08
1.4 ± 0.06
0.36
0.46
0.43
OC CRL (CaCl2)
2
OC CRL (MnCl )
3
4
4
64
65
*Activity (x10 )
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A
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B
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Time, (h)
Time, (h)
C
100
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0.5
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Time, (h)
Figure 1

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Time, (h)
Time, (h)
100
C
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Time, (h)
Figure 2

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00
Time, (h)
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Figure 3

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Time, (h)
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Figure 4

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Salt concentration, (mM)
Figure 5

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A
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60
80
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MgCl concentration, (mM)
2
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MgCl concentration, (mM)
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Figure 6

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Covalently
immobilized
RML or CRL
OC-support
interfacially
activated
RML or CRL
2
+
2+
Ca or Mn have not effect
on lipase stability
2+
2+
Ca or Mn produced a
significant
lipase stabilization
Free lipase