catalysts

Article
Covalent Immobilization of Candida rugosa Lipase
at Alkaline pH and Their Application in the
Regioselective Deprotection of
Per-O-acetylated Thymidine
Cintia W. Rivero † and Jose M. Palomo *
Departamento de Biocatálisis, Instituto de Catálisis (CSIC), Madrid 28049, Spain; [email protected]
* Correspondence: [email protected]; Tel.: +34-91-585-4768
† Current address: Laboratorio de Investigaciones en Biotecnología Sustentable (LIBioS),
Universidad Nacional de Quilmes, Roque Sáenz Peña 352, Bernal, Argentina

Academic Editor: Keith Hohn

Received: 10 June 2016; Accepted: 27 July 2016; Published: 2 August 2016

Abstract: Lipase from Candida rugosa (CRL) was stabilized at alkaline pH to overcome the inactivation
problem and was immobilized for the first time by multipoint covalent attachment on different
aldehyde-activated matrices. PEG was used as a stabilizing agent on the activity of CRL. At these
conditions, CRL maintained 50% activity at pH 10 after 17 h incubation in the presence of 40% (w/v)
of PEG, whereas the enzyme without additive was instantaneously inactive after incubation at pH
10. Thus, this enzyme was covalently immobilized at alkaline pH on three aldehyde-activated
supports: aldehyde-activated Sepharose, aldehyde-activated Lewatit105 and heterofunctional
aldehyde-activated EDA-Sepharose in high overall yields. Heterogeneous stable CRL catalysts
at high temperature and solvent were obtained. The aldehyde-activated Sepharose-CRL preparation
maintained 70% activity at 50 ˝ C or 30% (v/v) acetonitrile after 22 h and exhibited high regioselectivity
in the deprotection process of per-O-acetylated thymidine, producing the 31 -OH-51 -OAc-thymidine
in 91% yield at pH 5.

Keywords: Candida rugosa lipase; stabilization; covalent immobilization; PEG; alkaline pH;
regioselectivity; nucleosides

1. Introduction
Enzymes are natural catalysts working at mild conditions with high specificity by substrate and
excellent selectivity which could constitute a green solution for the industry. However, enzymes
are unstable out of their natural environments, and parameters such as pH and temperature (T) are
relevant to their stability. Therefore, the stabilization mechanism represents an important issue in
enzyme development for possible industrial implementation. Low enzyme stability has been overcome
using several strategies: genetic engineering [1], chemical modifications [2], the addition of stabilizing
agents [3–6], or the use of different immobilization mechanisms [7,8].
Particularly, multipoint covalent attachment on macroporous supports has been described as a
very interesting approach to stabilize enzymes [9–11].
The functionalization of the support materials by molecules as short spacer arms assures the
attachment of an enzyme molecule to the matrix through various covalent linkages. The amino-acidic
residues of the protein involved in the covalent immobilization should be rigid, conserving the relative
positions against changes in the protein conformation, e.g., with the effect of distorting agents, such as
heat and organic solvents [12–14].

Catalysts 2016, 6, 115; doi:10.3390/catal6080115 www.mdpi.com/journal/catalysts

Catalysts 2016, 6, 115 2 of 11

In this way the use of supports functionalized by aldehyde groups under alkaline conditions
promotes enzyme immobilization by a specific orientation, through the richest area containing the
highest number of lysines, generating a multipoint covalent interaction [15]. The advantages of the
aldehyde groups are: (i) they are reactive toward unprotonated primary amines; (ii) they are stable
under alkaline conditions; (iii) they do not exhibit steric hindrances for intramolecular reactions [16].
In fact, several industrial enzymes have been stabilized by multipoint covalent attachment on these
aldehyde-activated supports [15].
However, enzymes sensitive to alkaline pH are not able to be immobilized through this
methodology. Candida rugosa lipase (CRL), a very useful enzyme in biotransformations [17–19],
is one of these enzymes with very limited conditions for proper use, especially in regards to pH [20].
The immobilization methods for the covalent attachment of this lipase described in the literature
use the application of functionalized supports, e.g., with glutaraldehyde or cyanogen bromide
groups, for covalent immobilization at neutral pH, which mainly correspond to the reaction to the
terminal amino group, with a low, intense covalent immobilization [20,21]. Also, the adsorption
on hydrophobic supports at neutral pH has been used for improving its stability, although this is a
reversible immobilization and the enzyme can be leached from the support in the presence of some
concentration of solvent or detergent [20]. Therefore, the multipoint covalent immobilization of this
enzyme on aldehyde supports would be an excellent strategy, considering the advantages described,
for obtaining an irreversible, immobilized biocatalyst with high stability.
Improvements of the enzyme’s stability with the presence of some additives such as polyols,
solvents or sugars have been reported [22,23].
The use of these additives has the advantage of providing enzyme activity at high temperatures
and alkaline pH [24]. A study of the protective effect of PEG, trehalose and glycerol revealed an
increase with the reagent concentration and length of the carbon chain [25,26]. These compounds are
known to have a more or less pronounced effect on water activity and on the degree of water molecule
association [25]. On the other hand, aqueous solutions of polyols, polymers and sugars, as additives,
were used to study the thermostabilization of enzymes [27].
Herein, we propose a methodology to stabilize Candida rugosa lipase (CRL) at alkaline pH using
PEG as an additive, permitting for the first time its immobilization by multipoint covalent attachment
on aldehyde-activated (Ald) derivative supports. Two different covalent immobilization strategies
involving different orientations of the protein (Figure 1) in the immobilization were used (Scheme 1).
These new immobilized CRL biocatalysts were used to catalyze the regioselective monodeprotection
of per-O-acetylated thymidine, an interesting intermediate in the synthesis of different fungicidal,
antitumor, and especially antiviral agents [28].
 Herein, we propose a methodology to stabilize Candida rugosa lipase (CRL) at alkaline pH using 
PEG as an additive, permitting for the first time its immobilization by multipoint covalent attachment 
on aldehyde‐activated (Ald) derivative supports. Two different covalent immobilization strategies 
involving different orientations of the protein (Figure 1) in the immobilization were used (Scheme 1). 
These new immobilized CRL biocatalysts were used to catalyze the regioselective monodeprotection 
of per‐O‐acetylated thymidine, an interesting intermediate in the synthesis of different fungicidal, 
Catalysts 2016, 6, 115 3 of 11
antitumor, and especially antiviral agents [28]. 

A B

Ser209

Ser209

Catalysts 2016, 6, 115  3 of 11 
 
Figure 1. Structure of mature CRL. (A) Lid side. Oligopeptide lid (green), Lys (blue); (B) Lid opposite 
Figure 1. Structure of mature CRL. (A) Lid side. Oligopeptide lid (green), Lys (blue); (B) Lid opposite
side marked lysines; (C) Lid opposite side marked lysines and aspartic and alutamic acids (orange). 
side marked lysines; (C) Lid opposite side marked lysines and aspartic and alutamic acids (orange).
The structure of CRL was obtained from the Protein Data Bank (pdb code: CRL) and the picture was 
The structure of CRL was obtained from the Protein Data Bank (pdb code: CRL) and the picture was
created using Pymol v. 0.99. 
created using Pymol v. 0.99.

O
O H2
H C
O N
H2 H
O 1.Incubation pH 10, 
H C N
O 24 h, 4оC H
H2
O 2.NaBH4, 30 min, 4оC C
H N
+ H
Aldehyde-activated
Sepharose or H2N
Lewatit105 H2N Ald-CRL or
H2N Ald-Lewatit-CRL
H2N

H2N

O C. Rugosa Lipase (CRL) H2

O C N
H
O + 1.Incubation pH (ionic H2 H
O C N
H adsorption) H
O NH 3+ 2.Incubation pH 10,  O NH3+
N 24 h, 4оC N
H H
3.NaBH4, 30 min, 4оC
Aldehyde-EDA-activated
Sepharose Ald-EDA-CRL
 
Scheme 1. Covalent immobilization of CRL by different methodologies.
Scheme 1. Covalent immobilization of CRL by different methodologies. 

2. Results and Discussions

2. Results and Discussions 
2.1. Stabilization of CRL at Alkaline pH
2.1. Stabilization of CRL at Alkaline pH 
The stability of soluble pure CRL by incubation at different pHs was first studied (Figure 2). This
The stability of soluble pure CRL by incubation at different pHs was first studied (Figure 2). This 
lipase has been described to be a very sensitive enzyme [20]. After purification, the enzyme incubated
lipase has been described to be a very sensitive enzyme [20]. After purification, the enzyme incubated 
at pH 7 maintained around 92% activity during 25 h at 4 ˝ C. When the pH was increased up to 9,
at pH 7 maintained around 92% activity during 55 h at 4 °C. When the pH was increased up to 9, 60% 
60% activity was found after 25 h. However, the activity of the enzyme was totally lost immediately
activity was found after 55 h. However, the activity of the enzyme was totally lost immediately after 
after incubation at pH 10, exhibiting the extremely low stability at alkaline pH, which is mandatory to
incubation at  pH  10,  exhibiting  the  extremely low stability at  alkaline  pH,  which  is  mandatory  to 
perform the multipoint covalent immobilization on an aldehyde-activated support.
perform the multipoint covalent immobilization on an aldehyde‐activated support. 

100

80
ty (%)
 The stability of soluble pure CRL by incubation at different pHs was first studied (Figure 2). This 
lipase has been described to be a very sensitive enzyme [20]. After purification, the enzyme incubated 
at pH 7 maintained around 92% activity during 55 h at 4 °C. When the pH was increased up to 9, 60% 
activity was found after 55 h. However, the activity of the enzyme was totally lost immediately after 
incubation at  pH  10,  exhibiting  the  extremely low stability at  alkaline  pH,  which  is  mandatory  to 
Catalysts 2016, 6, 115 4 of 11
perform the multipoint covalent immobilization on an aldehyde‐activated support. 

100

80
Relative activity (%)

60

40

20

0
0 5 10 15 20 25 30

Time (h)  
Figure  2.  Stability 
Figure of  purified 
2. Stability CRL CRL
of purified at  25 
at °C 
25 ˝and  different 
C and pHs: 
different pHs:pH 
pH7  (rhombus), 
7 (rhombus),pH 
pH9 9(circles), 
(circles),pH 
pH 10 
104 of 11 
Catalysts 2016, 6, 115 
(squares); 0.27 mg purified free lipase was used in each experiment. 
(squares); 0.27 mg purified free lipase was used in each experiment.
Thus, different additives—at 20% (w/v) concentration—were added to the enzyme solution to 
study their effect on the enzyme stability (Figure 3). The best result was achieved when PEG was 
Thus, different additives—at 20% (w/v) concentration—were added to the enzyme solution to
previously 
study theiradded  to  the lipase alkaline 
effect on the enzyme stability solution (pH 
(Figure 3). 10), 
The where  the  enzyme 
best result retained 
was achieved when40% activity 
PEG was
after  incubation 
previously addedfor  4.5 lipase
to the h,  whereas 
alkalinethe  soluble 
solution (pHenzyme  without 
10), where additives 
the enzyme maintained 
retained only  20% 
40% activity after
activity. 
incubation Using  polyols 
for 4.5 such  as 
h, whereas theglycerol,  dextran  without
soluble enzyme or  trehalose,  a  slightly 
additives improved 
maintained only activity  was 
20% activity.
observed. 
Using polyols such as glycerol, dextran or trehalose, a slightly improved activity was observed.

100

80
Relative activity (%)

60

40

20

0
0 1 2 3 4 5

Time (h)
 
Figure 3. Stabilization of CRL in the presence of different additives. The additives were added at
Figure 3. Stabilization of CRL in the presence of different additives. The additives were added at 20% 
20% (w/v) concentration andexperiments 
the experiments were performed at pH ˝ C. Without additive
(w/v)  concentration  and  the  were  performed  at  pH  10 10 and4 4°C. 
and  Without  additive 
(squares), PEG (rhombus), glycerol (circles), dextran (triangles), threalose (ˆ).
(squares), PEG (rhombus), glycerol (circles), dextran (triangles), threalose (×). 

PEG 
PEG was 
was selected 
selected as 
as a 
a stabilizing 
stabilizing agent 
agent and 
and the 
the effect 
effect of 
of the 
the additive 
additive concentration 
concentration and 
and the 
the
molecular size on the lipase stability was studied (Figure 4). PEG1500 stabilized the enzyme slightly 
molecular size on the lipase stability was studied (Figure 4). PEG1500 stabilized the enzyme slightly
better than PEG6000 at a 15% concentration (w/v), although the best results were found using 40% 
better than PEG6000 at a 15% concentration (w/v), although the best results were found using 40%
(w/v) PEG1500, where CRL conserved 50% activity after 17 h incubation (Figure 4). 
(w/v) PEG1500, where CRL conserved 50% activity after 17 h incubation (Figure 4).

100

80
tive activity (%)

60
 PEG  was  selected  as  a  stabilizing  agent  and  the  effect  of  the  additive  concentration  and  the 
molecular size on the lipase stability was studied (Figure 4). PEG1500 stabilized the enzyme slightly 
better than PEG6000 at a 15% concentration (w/v), although the best results were found using 40% 
(w/v) PEG1500, where CRL conserved 50% activity after 17 h incubation (Figure 4). 
Catalysts 2016, 6, 115 5 of 11

100

80
Relative activity (%)

60

40

20

0
0 2 4 6 8 10 12 14 16 18

Time (h)  
Figure 4. Effect of PEG concentration on the stability of CRL at 4 ˝ C and pH 10. Without additive (ˆ),
Figure 4. Effect of PEG concentration on the stability of CRL at 4 °C and pH 10. Without additive (×), 
PEG-1500 15% (squares), PEG-6000 15% (circles), PEG-1500 20% (rhombus), PEG-1500 40% (triangles).
PEG‐1500 15% (squares), PEG‐6000 15% (circles), PEG‐1500 20% (rhombus), PEG‐1500 40% (triangles). 

The 
The possible 
possible mechanism 
mechanism for 
for the 
the high 
high stabilization 
stabilization of 
of lipase 
lipase achieved 
achieved by 
by the 
the addition 
addition of 
of PEG 
PEG
could be explained by two different effects. The first is (1) a strong physical adsorption of the PEG 
could be explained by two different effects. The first is (1) a strong physical adsorption of the PEG
molecules to the hydrophobic area of the protein, as previously has been reported [29]. Especially 
molecules to the hydrophobic area of the protein, as previously has been reported [29]. Especially
here, in lipases the most hydrophobic area is concentrated on the lid and the surrounding active site;
therefore, the PEG could generate protection of the active site, significantly improving the stability
in an extreme condition such as alkaline pHs. The second effect is (2) due to the fact that the use of
higher PEG concentrations causes a high viscosity which also may prevent undesired changes in the
enzyme structure promoted by the alkaline pH [30].

2.2. Multipoint Covalent Immobilization of CRL

The enzyme was immobilized at optimal conditions (with 40% PEG in solution) using two different
strategies to get multipoint covalent immobilization of the lipase throughout different orientations
(Scheme 1). CRL was immobilized on Ald-Sepharose at 89% yield (loading of 4.9 mg lipase/g
support) at pH 10.2 over 24 h retaining 53% initial activity, whereas a 69% yield of lipase immobilized
maintaining 39% of initial activity was obtained using Ald-Lew105 (Table 1).

Table 1. Covalent immobilization of Candida rugosa lipase on different supports at 4 ˝ C.

Support Immobilization yield (%) a Retained Activity (%)

Ald-Sepharose 89 53
Ald-Lew105 69 39
Ald-EDA-Sepharose 95 37
a Immobilization for 24 h; 5.5 mg pure lipase was offered per gram of support.

The immobilization on heterofunctional Ald-EDA-Sepharose was performed at pH 8 and around

90% yield was achieved after 3 h incubation. After that, the immobilized preparation was incubated at
pH 10 for 24 h to promote a possible multipoint covalent attachment. The immobilized preparation
retained 37% overall initial activity (Table 1).

2.3. Stability of Different Covalent Immobilized Preparations of CRL

To evaluate the effect of the immobilization method on the stabilization of the enzyme, inactivation
experiments at different conditions were studied (Figures 5 and 6).
Indeed, the Ald‐CRL preparation still conserved 70% of the initial activity after 22 h at 50 °C whereas 
the Ald‐Lew105‐CRL preparation only maintained 38% activity at this time. This demonstrates the 
effect of the matrix on the lipase stabilization. 
Also,  of  the  differently  oriented  catalysts,  Ald‐EDA‐CRL  showed  good  activity,  maintaining 
Catalysts 2016, 6, 115
around 60% activity after 22 h incubation (Figure 5).  6 of 11

100

80

Relative activity (%)

60

Catalysts 2016, 6, 115  40 6 of 11 

Figure 5. Thermal inactivation course of different CRL immobilized preparations. Experiments were 
20
performed  at  50  °C  and  pH  5.  Free  CRL  (×),  Ald‐EDA‐CRL  (rhombus),  Ald‐CRL  (squares),  Ald‐
Lew105‐CRL (circles). 
0
0 5 10 15 20 25
When the CRL preparations were incubated at 30% (v/v) acetonitrile at 25 °C, the effect was even 
Time (h)
clearer (Figure 6). The Ald‐CRL preparation was the most stable catalyst also in the presence of a co‐
 
solvent,  retaining  70%  activity  after  25  h  incubation,  conditions  where  the  soluble  enzyme  was 
Figure 5. Thermal inactivation course of different CRL immobilized preparations. Experiments
completely  inactive.  The ˝ enzyme  immobilized  on  the  Ald‐EDA  support  conserved  55%  activity 
were performed at 50 C and pH 5. Free CRL (ˆ), Ald-EDA-CRL (rhombus), Ald-CRL (squares),
whereas the Ald‐Lew105‐CRL preparation was again less stable than the Sepharose one (Figure 6). 
Ald-Lew105-CRL (circles).

100

80
Relative activity (%)

60

40

20

0
0 5 10 15 20 25

Time (h)  
Figure 6. Inactivation profile of different CRL immobilized preparations in the presence of co-solvent.
Figure 6. Inactivation profile of different CRL immobilized preparations in the presence of co‐solvent. 
Experiments were carried out at 25 ˝ C, pH 5 and 30% (v/v) acetonitrile. Free CRL (ˆ), Ald-EDA-CRL
Experiments were carried out at 25 °C, pH 5 and 30% (v/v) acetonitrile. Free CRL (×), Ald‐EDA‐CRL 
(rhombus), Ald-CRL (squares), Ald-Lew105-CRL (circles).
(rhombus), Ald‐CRL (squares), Ald‐Lew105‐CRL (circles). 

2.4. Regioselective Deprotection of Per‐O‐acetylated Thymidine by Immobilized CRL Biocatalysts 
At 50 ˝ C, the best stabilization of CRL was achieved after immobilization on Ald-Sepharose. The
Ald-CRL, Ald-lew105-CRL and Ald-EDA-CRL immobilized preparations conserved more than 50%
The different covalent immobilized preparations of CRL were used as catalysts in the hydrolytic 
activity after 9 h incubation at 50 ˝ C, whereas the free lipase only retained 6% activity (Figure 5).
deacetylation of per‐O‐acetylated thymidine (1) at different pHs (Table 2). 
Indeed,
The the Ald-CRLpreparation 
Ald‐CRL  preparationshowed 
still conserved 70% ofactivity 
the  highest  the initial activity
(three  after
times  at 50 ˝the 
22 h than 
higher  C whereas
soluble 
the Ald-Lew105-CRL preparation only maintained 38% activity at this time. This
enzyme) and regioselectivity in the monoacetylation of 1 at pH 5 and 7, producing the C‐3 hydroxy  demonstrates the
effect of the matrix on the lipase stabilization.
monoacetylated  thymidine  2  in  around  90%  yield.  The  other  CRL  preparations  showed  lower 
Also, of the and 
regioselectivity,  differently orientedin 
no  differences  catalysts,
activity Ald-EDA-CRL
compared  with  showed good enzyme 
the  soluble  activity, (Table 
maintaining
2).  In 
around 60% activity after 22 h incubation (Figure 5).
particular, CRL immobilized on Lew105 lost the specificity and the regioselectivity. Also, the results 
When the CRL preparations were incubated at 30% (v/v) acetonitrile at 25 ˝ C, the effect was
using the Ald‐CRL preparation at pH 8 were better than using the soluble enzyme (Table 2). The role 
even clearer (Figure 6). The Ald-CRL preparation was the most stable catalyst also in the presence
of the immobilization method on the modulation of the activity and regioselectivity has been shown. 
of a co-solvent, retaining 70% activity after 25 h incubation, conditions where the soluble enzyme
In the case of CRL, immobilization by this strategy generates a particular orientation of the enzyme 
was completely inactive. The enzyme
(from  the  protein  lid’s  opposite  side  immobilized on the
(Figure  1C))  and  Ald-EDA
a  strong  support conserved
rigidification  55% activity
of  its  structure.  This 
whereas the Ald-Lew105-CRL preparation was again less stable than the Sepharose
phenomenon alters the open‐closed movement of the oligopeptide lid during catalysis and the exact  one (Figure 6).
shape of the open structure which is translated in a significant modulation of the lipase properties. 
We  have  already  observed  these  alterations  in  lipase  enantioselectivity  [31],  and  also  in  the 
regioselective deprotection of different glycoderivatives [18], and it has been recently observed in the 
production of 2‐glyceryl derivatives [32]. 

Table 2. Regioselective deprotection of 3,5‐O‐diacetylated thymidine 1 with different aldehyde‐activated 
Catalysts 2016, 6, 115 7 of 11

2.4. Regioselective Deprotection of Per-O-acetylated Thymidine by Immobilized CRL Biocatalysts

The different covalent immobilized preparations of CRL were used as catalysts in the hydrolytic
deacetylation of per-O-acetylated thymidine (1) at different pHs (Table 2).

Table 2.
Catalysts 2016, 6, 115  Regioselective deprotection of 3,5-O-diacetylated thymidine 1 with different7 of 11 
aldehyde-activated CRL biocatalysts at 25 ˝ C in aqueous media.
O O O

NH NH NH

N O N O N O
AcO AcO HO
O O O
+
CRL-biocatalysts OAc
OAc OH
1 2 3  
Initial a Reaction  Yield b b
Reaction
Biocatalyst 
BiocatalystpH pHInitial Rate 
a
Yield   (%)
2 (%) 2 (%)
3 (%) 3 (%)  Thymidine
Thymidine
Rate Time (h)
Time (h) (%)
free CRL 
free CRL 5.0 5.0 0.08 
0.08 104 
104 99 99  81 9
81 9  10 10 
Ald‐CRL Ald-CRL 5.0 5.0 0.24 
0.24 71 
71 100 100  91 3
91 3  6 6 
Ald-EDA-CRL
Ald‐EDA‐CRL  5.0 5.0 0.09
0.09  144
144  100 100  70
70 10 10  20 20 
Lew105-CRL 5.0 0.06 144 100 17 15 67
Lew105‐CRL  5.0 0.06  144  100  17  15  67 
free CRL 7.0 0.08 104 99 90 8 2
free CRL 
Ald-CRL 7.0 7.0 0.08 
0.26 104 
51 100 99  90 4
90 8  6 2 
Ald‐CRL Ald-EDA 7.0 7.0 0.26 
0.09 51 
152 100 100  90 10
88 4  3 6 
Ald‐EDA  Lew105 7.0 7.0 0.04
0.09  150
152  62 100  28
88 32 10  2 3 
Lew105 free CRL 7.0 8.0 0.08
0.04  120
150  100 62  75
28 11 32  13 2 
Ald-CRL 8.0 0.11 73 100 88 8 4
free CRL  8.0 0.08  120  100  75  11  13 
a the initial rate in µmol ˆ mgprot´1 ˆ h´1 . It was calculated at 10%–30% conversion. b yield of the
Ald‐CRL  8.0 0.11  73 
monohydroxy acetylated product at 100% conversion.
100  88  8  4 
  the  initial  rate  in  μmol  ×  mgprot−1  ×  h−1.  It  was  calculated  at  10%–30%  conversion.  b  yield  of  the 
a

monohydroxy acetylated product at 100% conversion. 
The Ald-CRL preparation showed the highest activity (three times higher than the soluble
enzyme) and regioselectivity in the monoacetylation of 1 at pH 5 and 7, producing the C-3 hydroxy
3. Experimental Section 
monoacetylated thymidine 2 in around 90% yield. The other CRL preparations showed lower
regioselectivity, and no differences in activity compared with the soluble enzyme (Table 2). In particular,
3.1. Materials 
CRL immobilized on Lew105 lost the specificity and the regioselectivity. Also, the results using the
Lipase 
Ald-CRL from  Candida 
preparation at pHrugosa 
8 were(CRL), 
betterethylendiamine  (EDA), enzyme
than using the soluble p‐nitrophenyl  butyrate 
(Table 2). (pNPB), 
The role of the
dithiothreitol 
immobilization (DTT), 
methodpolyethyleneglycol 
on the modulation(PEG) 
of the(Mr  1500, 
activity 6000), 
and glycerol,  dextran 
regioselectivity has been(Mw  1500) 
shown. Inand 
the
trehalose were from Sigma Chem. Co (St. Louis, MO, USA). Sepharose 10BCL, octyl‐Sepharose and 
case of CRL, immobilization by this strategy generates a particular orientation of the enzyme (from the
cyanogen bromide (CNBr) activated Sepharose beads were from GE‐Healthcare (Uppsala, Sweden). 
protein lid’s opposite side (Figure 1C)) and a strong rigidification of its structure. This phenomenon
Aldehyde‐activated 
alters the open-closed Sepharose 
movementor ofLewatit  VP  OC105 
the oligopeptide (Ald  or 
lid during Ald‐Lew105) 
catalysis and thewere 
exactprepared  as 
shape of the
previously described [33]. 3,5‐O‐diacetylated thymidine 1 was prepared as previously described [34]. 
open structure which is translated in a significant modulation of the lipase properties. We have already
observed these alterations in lipase enantioselectivity [31], and also in the regioselective deprotection
3.2. Lipase Activity Assay 
of different glycoderivatives [18], and it has been recently observed in the production of 2-glyceryl
derivatives [32].
The  activities  of  the  soluble  lipase  (without  additives  or  in  the  presence  of  different 
concentrations  of  PEG,  glycerol,  DTT,  trehalose  or  dextran),  supernatant  and  enzyme  suspension 
3. Experimental Section
were analyzed spectrophotometrically measuring the increment in absorbance at 348 nm produced 
by the release of p‐nitrophenol (pNP) (Є = 5.150 M
3.1. Materials
−1∙cm−1) in the hydrolysis of 0.4 mM pNPB in 25 

mM sodium phosphate at pH 7 and 25 °C. To initialize the reaction, 0.05–0.2 mL of lipase solution or 
Lipase from Candida rugosa (CRL), ethylendiamine (EDA), p-nitrophenyl butyrate (pNPB),
suspension was added to 2.5 mL of substrate solution in magnetic stirring. Enzymatic activity is given 
dithiothreitol (DTT),
as  one  μmol  of  polyethyleneglycol
p‐nitrophenol  (PEG)
released  per  (Mr per 
minute  1500, 6000),
mg  glycerol,
of  enzyme  dextran
(IU)  under (Mw 1500) and
the  conditions 
trehalose were from
described above.  Sigma Chem. Co (St. Louis, MO, USA). Sepharose 10BCL, octyl-Sepharose and
cyanogen bromide (CNBr) activated Sepharose beads were from GE-Healthcare (Uppsala, Sweden).
3.3. C. rugosa Lipase Purification 
The enzyme was purified from commercial crude extract by interfacial adsorption as previously 
described [35]. Lipase commercial extract was dissolved in 25 mM sodium phosphate buffer at pH 7 
to give 200 mg extract/mL, and submitted to gentle stirring during 1 h at 4 °C, and centrifuged at 
12,000 rpm during 15 min. The supernatant was separated from the pellet and the protein amount 
Catalysts 2016, 6, 115 8 of 11

Aldehyde-activated Sepharose or Lewatit VP OC105 (Ald or Ald-Lew105) were prepared as previously

described [33]. 3,5-O-diacetylated thymidine 1 was prepared as previously described [34].

3.2. Lipase Activity Assay

The activities of the soluble lipase (without additives or in the presence of different concentrations
of PEG, glycerol, DTT, trehalose or dextran), supernatant and enzyme suspension were analyzed
spectrophotometrically measuring the increment in absorbance at 348 nm produced by the release
of p-nitrophenol (pNP) (Є= 5.150 M´1 ¨cm´1 ) in the hydrolysis of 0.4 mM pNPB in 25 mM sodium
phosphate at pH 7 and 25 ˝ C. To initialize the reaction, 0.05–0.2 mL of lipase solution or suspension
was added to 2.5 mL of substrate solution in magnetic stirring. Enzymatic activity is given as one µmol
of p-nitrophenol released per minute per mg of enzyme (IU) under the conditions described above.

3.3. C. rugosa Lipase Purification

The enzyme was purified from commercial crude extract by interfacial adsorption as previously
described [35]. Lipase commercial extract was dissolved in 25 mM sodium phosphate buffer at pH 7
to give 200 mg extract/mL, and submitted to gentle stirring during 1 h at 4 ˝ C, and centrifuged at
12,000 rpm during 15 min. The supernatant was separated from the pellet and the protein amount was
calculated by Bradford method [36] (5.5 mg prot/mL). Then 1 mL of this supernatant was diluted in
9 mL of 25 mM phosphate buffer pH 7 and the solution was added to one gram of octyl-Sepharose.
The reaction was performed at 4 ˝ C for 1 h. After that, the suspension was filtered by vacuum and the
solid was washed several times with distilled water. More than 95% of the enzyme was immobilized.
For the preparation of the covalent immobilized catalysts, the lipase was desorbed from the
support (one gram of octyl-CRL) adding 20 mL of a solution of 25 mM phosphate buffer pH 7 with
0.4% Triton X-100 (v/v) and incubated it for 1 h. A final solution of 0.27 mg purified lipase/mL
was obtained.

3.4. Preparation of EDA-Aldehyde–Activated Sepharose Support (Ald-EDA)

Sepharose 10 BCL (10 g) was suspended in a mixture solution of 44 mL water, 16 mL acetone,
3.28 g NaOH, 0.2 g NaBH4 and 11 mL epichlorhydrine. The suspension was stirred mildly for 16 h and
washed with an excess of water. One gram of epoxy-Sepharose support was suspended in 10 mL of
ethylenediamine (0.1 M) solution at pH 8 for 6 h. Finally the support was oxidized adding a solution
of 10 mL of water with 140 µmol of sodium periodate per gram of support during 90 minutes and
washed abundantly with distilled water and store at 4 ˝ C.

3.5. Multipoint Covalent Immobilization of CRL on Different Aldehyde-Activated Sepharose Supports (Ald)

3.5.1. Immobilization on Aldehyde-Activated Sepharose (Ald) or Aldehyde-Activated Lewatit-105

(Ald-Lew105) at Alkaline pH
First 20 mL of lipase solution (0.27 mglipase /mL) was dissolved in 20 mL solution of 100 mM
sodium bicarbonate pH 8.2 containing 40% PEG1500 (w/v) and the pH was adjusted at pH 10.15. After
that, one gram of Ald-Sepharose or Ald-Lew-105 was added and the reaction was maintained during
16 h at 4 ˝ C. Finally the enzyme-support multi-interaction was ended by adding 1 mg of sodium
borohydride per mL of suspension during 30 min [33] (Scheme 1). The immobilization yields are
shown in Table 1.

3.5.2. Immobilization on Aldehyde-Activated EDA-Sepharose (Ald-EDA) at pH 8 and Incubation

at pH 10
One gram of Ald-EDA-Sepharose was added to 10 mL of purified CRL solution (0.27 mg lip/mL)
containing 40% PEG1500 (w/v). Then the suspension was stirred for 2 h at pH 8 and 4 ˝ C. Periodically,
samples of the supernatants and suspensions were withdrawn, and the enzyme activity was measured
Catalysts 2016, 6, 115 9 of 11

as described above. After the preparation was filtrated by vacuum and the solid was incubated in
10 mL sodium bicarbonate buffer at pH 10 for 24 h. Finally, the preparation was reduced by addition of
10 mg sodium borohydride for 30 minutes and then washed with water (Scheme 1). The immobilization
yields are shown in Table 1.

3.6. Inactivation of CRL Immobilized Preparations against T and Co-Solvent

First 0.5 g of biocatalyst were dissolved in 5 mL of 25 mM sodium phosphate buffer (with 30%
(v/v) acetonitrile) at 25 ˝ C or incubated at 50 ˝ C in acetate buffer at pH 5. The remaining activity at
different times was measured by the assay described above using pNPB as substrate.

3.7. Enzymatic Hydrolysis of 31 ,51 -Di-O-Acetylthymidine (1)

Substrate 1 (5 mM) was dissolved in a mixture of acetonitrile (5%, v/v) in 10 mM sodium
phosphate at pH 7.0 or 10 mM sodium acetate at pH 5.0. 0.2 g of biocatalyst was added to 2 mL of this
solution at 25 ˝ C. During the reaction, the temperature and the pH value was maintained constant
using a pH-stat Mettler Toledo DL50 graphic (Mettler-Toledo, LLC 1900 Polaris Parkway, Columbus,
OH, USA). The degree of hydrolysis was analyzed by reverse phase HPLC (Spectra Physic Thermo
SP 100 coupled with an UV detector Spectra Physic SP 8450 (Thermo Fisher-Scientific, Waltham, MA,
USA). For these assays a Kromasil C18 5 µm ϕ (25 cmˆ0.4 cm) column was used and the following
gradient program (A: mixture of acetonitrile (10%, v/v) in 10mM ammonium phosphate at pH 4.2; B:
mixture of miliQ water (10%, v/v) in acetonitrile; method: 0–6 min 100% A, 6–14 min 85% A to 15%B,
14–22 min 100% A, flow: 1.0 mL¨min´1 ). UV detection was performed at 260 nm. The unit of enzymatic
activity was defined as micromoles of substrate hydrolyzed per minute per mg of immobilized protein.
The monodeprotected 3-OH (2) and 5-OH (3) were used as pure standards. The retention time was
2.4 min for Thymidine, 9.4 min for 2 and 10.2 min 5 and 19 min for 1.

4. Conclusions
Lipase from C. rugosa has been stabilized at alkaline pH to overcome the inactivation problem
by the addition of PEG1500 as a stabilizing agent. Therefore, this has permitted its immobilization
for the first time by multipoint covalent attachment on different aldehyde-activated supports in high
overall yields. Very stable CRL biocatalysts have been prepared; in particular CRL immobilized on
Ald-Sepharose was much more stable than the soluble enzyme. This stable biocatalyst showed
an excellent regioselectivity in the monodeprotection of per-O-acetylated thymidine, producing
the 3-OH-51 -OAc-thymidine in 91% yield at pH 5. Therefore, this new biocatalyst represents an
interesting alternative to the octyl-CRL preparation, which has been described as an excellent catalyst
in nucleosides and especially in monosaccharides deprotection [18]. However, Ald-CRL presents the
advantage of being an irreversible catalyst with high stability in the presence of solvent. This strategy
can be also extended to other pH-sensitive enzymes to generate highly stable and active biocatalysts.

Acknowledgments: This work has been sponsored by CSIC. The authors gratefully recognize the support from
National Scientific and Technical Research Council (CONICET).
Author Contributions: J.M.P. conceived and designed the experiments; C.W.R. performed the experiments; J.M.P.
wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Reetz, M.T. Biocatalysis in Organic Chemistry and Biotechnology: Past, Present, and Future. J. Am. Chem. Soc.
2013, 135, 12480–12496. [CrossRef] [PubMed]
2. Xue, Y.; Wu, C.-Y.; Branford-White, C.J.; Ning, X.; Nie, H.-L.; Zhu, L.-M. Chemical modification of stem
bromelain with anhydride groups to enhance its stability and catalytic activity. J. Mol. Catal. B Enzym. 2010,
63, 188–193. [CrossRef]
Catalysts 2016, 6, 115 10 of 11

3. Srirangsan, P.; Kawai, K.; Hamada-Sato, N.; Watanabe, M.; Suzuki, T. Stabilizing effects of sucrose-polymer
formulations on the activities of freeze-dried enzyme mixtures of alkaline phosphatase, nucleoside
phosphorylase and xanthine oxidase. Food Chem. 2011, 125, 1188–1193. [CrossRef]
4. Santagapita, P.R.; Brizuela, L.G.; Mazzobre, M.F.; Ramírez, H.L.; Corti, H.R.; Santana, R.V.; Buera, M.P.
β-cyclodextrin modifications as related to enzyme stability in dehydrated systems: Supramolecular
transitions and molecular interactions. Carbohydr. Polym. 2011, 83, 203–209. [CrossRef]
5. Nascimento, C.; Leandro, J.; Lino, P.R.; Ramos, L.; Almeida, A.J.; De Almeida, I.T.; Leandro, P. Polyol
additives modulate the in vitro stability and activity of recombinant human phenylalanine hydroxylase.
Appl. Biochem. Biotechnol. 2010, 162, 192–207. [CrossRef] [PubMed]
6. Iyer, P.V.; Ananthanarayan, L. Enzyme stability and stabilization-Aqueous and non-aqueous environment.
Process Biochem. 2008, 43, 1019–1032. [CrossRef]
7. Guisán, J.M. Immobilization of Enzymes and Cells, Methods in Molecular Biology, 3rd ed.; Humana Press:
New York, NY, USA, 2013; pp. 1–377.
8. Mateo, C.; Grazu, V.; Palomo, J.M.; Lopez-Gallego, F.; Fernandez-Lafuente, R.; Guisán, J.M. Immobilization
of Enzymes on Heterofunctional Epoxy Supports. Nat. Protoc. 2007, 2, 1022–1033. [CrossRef] [PubMed]
9. Katchalski-Katzir, E. Immobilized enzymes—Learning from past successes and failures. Trends Biotechnol.
1993, 11, 471–478. [CrossRef]
10. Cao, L. Immobilised enzymes: Science or art? Curr. Opin. Chem. Biol. 2005, 9, 217–226. [CrossRef] [PubMed]
11. Mateo, C.; Palomo, J.M.; Fernandez-Lorente, G.; Guisan, J.M.; Fernandez-Lafuente, R. Improvement of
enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol. 2007, 40,
1451–1463. [CrossRef]
12. Pedroche, J.; Yust, M.M.; Mateo, C.; Fernández-Lafuente, R.; Girón-Calle, J.; Alaiz, M.; Vioque, J.; Guisan, J.M.;
Millan, F. Effect of the support and experimental conditions in the intensity of the multipoint covalent
attachment of proteins on aldehyde activated-Sepharose supports: Correlation between enzyme–support
linkages and thermal stability. Enzyme Microb. Technol. 2007, 40, 1160–1167. [CrossRef]
13. Bolivar, J.M.; Rocha-Martin, J.; Mateo, C.; Cava, F.; Berenguer, J.; Vega, D.; Fernandez-Lafuente, R.;
Guisan, J.M. Purification and stabilization of a glutamate dehygrogenase from Thermus thermophilus
via oriented multisubunit plus multipoint covalent immobilization. J. Mol. Catal. B Enzym. 2009, 58, 158–163.
[CrossRef]
14. Klibanov, A.M. Stabilization of Enzymes against Thermal Inactivation. Adv. Appl. Microbiol. 1983, 29, 1–28.
[PubMed]
15. Mateo, C.; Abian, O.; Bernedo, M.; Cuenca, E.; Fuentes, M.; Fernandez-Lorente, G.; Palomo, J.M.; Grazu, V.;
Pessela, B.C.C.; Giacomini, C.; et al. Some special features of aldehyde activated supports to immobilize
proteins. Enzyme Microb. Technol. 2005, 37, 456–462. [CrossRef]
16. Blanco, R.M.; Calvete, J.J.; Guisan, J.M. Immobilization-stabilization of enzymes; variables that control the
intensity of the trypsin (amine)-Sepharose (aldehyde) multipoint attachment. Enzyme Microb. Technol. 1989,
11, 353–359. [CrossRef]
17. Kolodiazhnyi, O.I. Recent developments in the asymmetric synthesis of -chiral phosphorus compounds.
Tetrahedron Asymmetry 2012, 23, 1–46.
18. Filice, M.; Guisan, J.M.; Terreni, M.; Palomo, J.M. Regioselective monodeprotection of peracetylated
carbohydrates. Nat. Protoc. 2012, 7, 1783–1796. [CrossRef] [PubMed]
19. Domínguez de María, P.; Alcántara, A.R.; Carballeira, J.D.; de la Casa, R.M.; García-Burgos, C.A.;
Hernáiz, M.J.; Sánchez-Montero, J.M.; Sinisterra, J.V. Candida rugosa Lipase: A traditional and complex
biocatalyst. Curr. Org. Chem. 2006, 10, 1053–1066. [CrossRef]
20. Palomo, J.M.; Fernández-Lorente, G.; Mateo, C.; Ortiz, C.; Fernandez-Lafuente, R.; Guisan, J.M. Modulation
of the enantioselectivity of lipases via controlled immobilization and medium engineering Hydrolytic
resolution of Mandelic acid esters. Enzyme Microb. Technol. 2002, 31, 775–783. [CrossRef]
21. Knezevic, Z.; Milosavic, N.; Bezbradica, D.; Jakovljevic, Z.; Prodanovic, R. Immobilization of lipase from
Candida rugosa on Eupergit® C supports by covalent attachment. Biochem. Eng. J. 2006, 30, 269–278.
[CrossRef]
22. Noriko, M.; Miok, K.; Tadao, K. Stabilization of L-Ascorbic acid by superoxide dismutase and catalase.
Biosci. Biotechnol. Biochem. 1999, 63, 54–57.
Catalysts 2016, 6, 115 11 of 11

23. Lozano, P.; Combes, D.; Iborra, J.L. Effects of polyols on chymotrypsin thermostability a mechanistic analysis
of the enzyme stabilization. J. Biotechnol. 1994, 35, 9–18. [CrossRef]
24. Castro, G.R. Properties of soluble α-chymotrypsin in neat glycerol and water. Enzyme Microb. Technol. 2000,
27, 143–150. [CrossRef]
25. Combes, D.; Yoodikhya, T.; Girbal, E.; Willemot, R.M.; Monsan, P. Mechanism of Enzyme Stabilization.
Ann. N. Y. Acad. Sci. 1987, 501, 59–62. [CrossRef] [PubMed]
26. Salahas, G.; Peslis, B.; Georgiou, C.D.; Gavalas, N.A. Trehalose, an extreme temperature protector of
phosphoenolpyruvate carboxylase from the C4-plant Cynodon dactylon. Phytochemistry 1997, 46, 1331–1334.
[CrossRef]
27. Breccia, J.D.; Moran, A.C.; Castro, G.R.; Siñeriz, F. Thermal stabilization by polyols of β-xylanase from
Bacillus amyloliquefaciens. J. Chem. Technol. Biotechnol. 1998, 71, 241–245. [CrossRef]
28. Jordheim, L.P.; Durantel, D.; Zoulim, F.; Dumontet, C. Advances in the development of nucleoside and
nucleotide analogues for cancer and viral diseases. Nat. Rev. Drug Discov. 2013, 12, 447–464. [CrossRef]
[PubMed]
29. Rawat, S.; Raman Suri, C.; Sahoo, D.K. Molecular mechanism of polyethylene glycol mediated stabilization
of protein. Biochem. Biophys. Res. Commun. 2010, 392, 561–566. [CrossRef] [PubMed]
30. Fernández-Lorente, G.; Lopez-Gallego, F.; Bolivar, J.M.; Rocha-Martin, J.; Moreno-Perez, S.; Guisan, J.M.
Immobilization of proteins on highly activated glyoxyl supports: Dramatic increase of the enzyme stability
via multipoint immobilization on pre-existing carriers. Curr. Org. Chem. 2015, 19, 1719–1731. [CrossRef]
31. Palomo, J.M. Lipases Enantioselectivity Alteration by Immobilization Techniques. Curr. Biol. Compet. 2008, 4,
126–138. [CrossRef]
32. Guajardo, N.; Bernal, C.; Wilson, L.; Cabrera, Z. Selectivity of R-α-monobenzoate glycerol synthesis catalyzed
by Candida antarctica lipase B immobilized on heterofunctional supports. Process Biochem. 2015, 50,
1870–1877. [CrossRef]
33. Mateo, C.; Palomo, J.M.; Fuentes, M.; Betancor, L.; Grazu, V.; Lopez-Gallego, F.; Pessela, B.C.C.; Hidalgo, A.;
Fernandez-lorente, G.; Fernandez-lafuente, R.; et al. Glyoxyl agarose: A fully inert and hydrophilic support
for immobilization and high stabilization of proteins. Enzyme Microb. Technol. 2006, 39, 274–280. [CrossRef]
34. Romero, O.; Filice, M.; de Las Rivas, B.; Carrasco-Lopez, C.; Klett, J.; Morreale, A.; Hermoso, J.A.; Guisan, J.M.;
Abian, O.; Palomo, J.M. Semisynthetic peptide-lipase conjugates for improved biotransformations.
Chem. Commun. 2012, 48, 9053–9055. [CrossRef] [PubMed]
35. Bastida, A.; Sabuquillo, P.; Armisen, P.; Fernandez-Lafuente, R.; Huguet, J.; Guisan, J.M. A single
step purification, immobilization, and hyperactivation of lipases via interfacial adsorption on strongly
hydrophobic supports. Biotechnol. Bioeng. 1998, 58, 486–493. [CrossRef]
36. Bradford, M.M. A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing
the principle of protein dye binding. Anal. Biochem. 1976, 72, 248–254. [CrossRef]

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