Improving enantioselectivity of lipase from Candida rugosa by carrier-bound and carrier-free immobilization

Article abstract of DOI:10.1016/j.molcatb.2016.04.006

The enantioselectivity of carrier-bound and carrier-free immobilized lipase from Candida rugosa (CRL) was studied. CRL was immobilized in six agarose-based carriers functionalized with different reactive groups and in two different CRL cross-linked aggregates. Both, activity and enantioselectivity of all the immobilized lipase preparations were evaluated with different racemic esters under different reaction conditions (temperature, pH and solvent polarity). A strong effect of reaction media and immobilization protocol on enzyme activity and selectivity was found. Enzyme immobilization and reaction engineering allowed us obtaining the best immobilization protocol and reaction conditions to achieve high activity and enantioselectivty of CRL as heterogeneous catalyst. CRL immobilized on an agarose-based carrier activated with primary amino groups preferentially hydrolyzed (S)-phenylethyl acetate with E > 200 under pH 7, 4 °C and 30% of acetonitrile. On the other hand, CRL aggregated and cross-linked through their carboxylic groups preferentially hydrolyzed the (S)-isomer of ethyl 2-hydroxy-4-phenylbutyrate with an E = 39 under pH 5, 4 °C and 30% of acetonitrile. This work demonstrates the success of the combinatorial enzyme engineering for the production of highly enantioselective heterogeneous biocatalysts by screening different immobilization protocols and reaction media conditions.

Full text of DOI:10.1016/j.molcatb.2016.04.006

Journal of Molecular Catalysis B: Enzymatic 130 (2016) 32–39
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Improving enantioselectivity of lipase from Candida rugosa by
carrier-bound and carrier-free immobilization
Susana Velasco-Lozanoa,b, Fernando López-Gallego , Javier Rocha-Martin ,
José Manuel Guisán , Ernesto Favela-Torres
a
b
c
d,∗
a,∗
Departamento de Biotecnología Universidad Autónoma Metropolitana Iztapalapa. Av. San Rafael Atlixco #186, Col. Vicentina 09340, D.F., Mexico
Heterogeneous biocatalysis group, CIC Biomagune, Parque tecnológico de San Sebastián, Edificio Empresarial “C”, Paseo Miramón 182, 20009,
b
Donostia-San Sebastián Guipúzcoa, Spain
c
Abengoa Research S. L. Campus Palmas Altas, 41014, Seville, Spain
Departamento de Biocatálisis, Instituto de Catálisis (CSIC), Campus UAM Cantoblanco, 28049, Madrid, Spain
d
a r t i c l e i n f o
a b s t r a c t
Article history:
The enantioselectivity of carrier-bound and carrier-free immobilized lipase from Candida rugosa (CRL)
was studied. CRL was immobilized in six agarose-based carriers functionalized with different reactive
groups and in two different CRL cross-linked aggregates. Both, activity and enantioselectivity of all the
immobilized lipase preparations were evaluated with different racemic esters under different reaction
conditions (temperature, pH and solvent polarity). A strong effect of reaction media and immobilization
protocol on enzyme activity and selectivity was found. Enzyme immobilization and reaction engineering
allowed us obtaining the best immobilization protocol and reaction conditions to achieve high activity
and enantioselectivty of CRL as heterogeneous catalyst. CRL immobilized on an agarose-based carrier acti-
vated with primary amino groups preferentially hydrolyzed (S)-phenylethyl acetate with E > 200 under
Received 21 January 2016
Received in revised form 8 April 2016
Accepted 8 April 2016
Available online 12 May 2016
Keywords:
Immobilization
Enantioselectivity
CLEA
◦
Lipase
pH 7, 4 C and 30% of acetonitrile. On the other hand, CRL aggregated and cross-linked through their
carboxylic groups preferentially hydrolyzed the (S)-isomer of ethyl 2-hydroxy-4-phenylbutyrate with
◦
an E = 39 under pH 5, 4 C and 30% of acetonitrile. This work demonstrates the success of the combina-
torial enzyme engineering for the production of highly enantioselective heterogeneous biocatalysts by
screening different immobilization protocols and reaction media conditions.
©
2016 Elsevier B.V. All rights reserved.
1
. Introduction
such as temperature [9], presence of co-solvents [10] and additives
11,12], substrate concentration [13,14], water [15], etc. In the last
[
Enantioselective biotechnological processes have gained inter-
decade, enzyme immobilization has been revealed as a powerful
technique to enhance enzyme enantioselectivity beyond its capac-
ity to turn enzyme into re-usable and stable biocatalysts [16–20].
Therefore, an optimal combination of immobilization techniques
and reaction conditions allows easy, fast and low cost preparation
of enantioselective heterogeneous biocatalysts.
There are many classifications for immobilization protocol;
one of the most frequent is based on whether the insoluble
enzyme is either carrier-bound or carrier-free. Among carrier-
bound techniques, there are diverse types of carriers driving
enzyme immobilization by adsorption, entrapment, encapsula-
tion or covalent-binding on pre-existing matrixes [21]. Carrier-free
immobilization is based on the formation of protein-protein aggre-
gates covalently cross-linked (CLEA). Carrier-free immobilization
is a less expensive option since no matrix cost contributes to the
biocatalyst price. Moreover, CLEA technology yields biocatalysts
with higher volumetric productivities because the major part of
est because of exquisite enzyme selectivity [1]. The growing global
market of chiral technology was estimated in $5.3 billion dollars in
the year 2011, and its projection for 2016 is nearly $6.5 billion dol-
lars [2]. In this scenario, enzymes have been intensively exploited
in kinetic resolution and asymmetric synthesis of optically pure
compounds with high impact on pharmaceutical, agricultural and
food industries [3–6].
Enzyme enantioselectivity is a multi-variable dependent cat-
alytic feature which can be altered either by directly engineering
the biocatalysts (protein engineering or enzyme immobilization)
[
7,8] or by reaction engineering controlling different conditions
∗ Corresponding authors.
E-mail addresses: jmguisan@icp.csic.es (J.M. Guisán), favela@xanum.uam.mx
E. Favela-Torres).
(
http://dx.doi.org/10.1016/j.molcatb.2016.04.006
381-1177/© 2016 Elsevier B.V. All rights reserved.
1

S. Velasco-Lozano et al. / Journal of Molecular Catalysis B: Enzymatic 130 (2016) 32–39
33
3. Methods
3.1. Carrier-free immobilization of CRL
CRL was immobilized as carboxyl-activated cross-linked
enzyme aggregates (c-CLEA) and by amino cross-linked enzyme
aggregates (a-CLEA) as described previously [29]. EDC-NHS 28 mM
and GA 14 mM were employed for c-CLEA and a-CLEA respectively.
In both cases, PEI at 3 g/L and 10 mg of BSA as protein feeder were
employed.
3
3
.2. Carrier-bound immobilization
Scheme 1. (A) Enantioselective hydrolysis of rac-1 for the lipase catalyzed res-
olution of (S)-2. (B) Lipase catalyzed enantioselective hydrolisis of rac-3 for the
resolution of (R)-3.
.2.1. Immobilization of CRL on cyanogen bromide activated
agarose (CNBr-CRL)
According with a previous report [30], the immobilization pro-
cedure was conducted by mixing 10:1 (v/w) the enzyme solution
(
7
1 mg of enzyme powder per mL of 25 mM phosphate buffer pH
insoluble biocatalyst mass belongs to the enzyme [22]. However,
to select the best immobilization protocol for a certain enzyme,
both carrier-free and carrier bound must be tested in order to fit
the best biocatalyst properties to the optimal chemical process
configuration.
, 0.05% w/v Triton® X-100) with CNBr-activated agarose (deter-
gent avoids the attachment of lipase dimmers [31]) and maintained
under gentle agitation at 4 C for 7 min. The immobilization was
stopped by filtration (30% of enzyme was loaded to the support)
and 10 mL of ethanolamine 1 M at pH 7 were added and let them
to react 2 h at 4 C. Then, the immobilized preparation was filtered
◦
In this work, activity and enantioselectivity of Candida rugosa
lipase (CRL) were evaluated after combinatorial screening of both
immobilization protocols and reaction media conditions. A sur-
vey of immobilized biocatalysts was assayed towards different
esters under different reaction conditions controlling tempera-
ture, pH and media polarity. As model chiral reactions, two esters
with different acyl substituents; an alcohol and hydroxyacid ester
were evaluated for enantioselective hydrolysis (Scheme 1). Chiral
alcohols and ␣-hydroxyacids play an important role as building
blocks in fine chemistry [23–25]. Therefore, we studied the enan-
tioselectivity of different CRL derivatives towards the resolution
of (S)-1-phenylethanol (S-2) starting from racemic 1-phenylethyl
acetate (rac-1). Besides, we also tested the steroselective hydroly-
sis of racemic ethyl 2-hydroxy-4-phenylbutyrate (rac-3) to yield
ethyl (R)-2-hydroxy-4-phenylbutyrate (R-3); a key precursor in
the synthesis of angiotensin-converterting enzyme inhibitors [26]
like enalapril, benazepril, cilazapril, ramipril and quinapril applied
in the treatment of hypertension and heart diseases [27,28]. This
study is one of the scarcely works which compares the performance
of carrier-free and carrier-bound heterogeneous lipases towards
different enantioselective resolutions.
◦
and washed with 10 vol of water and finally rinsed with 25 mM
phosphate buffer pH 7 and stored at 4 C.
◦
3
.2.2. Immobilization of CRL on octyl-sepharose (octyl-CRL)
Based in a previous described methodology [32,33], hydropho-
bic adsorption of CRL on octyl-sepharose was carried out as follows:
ten millilitres of enzyme solution (1 mg of enzyme powder per mL
of 25 mM phosphate buffer pH 7) were mixed with 1 g of octyl-
◦
sepharose and were maintained under gentle agitation at 25 C. The
immobilization curse was followed by measuring the activity in the
supernatant and in the suspension every 30 min. Once no activity
was detected in the supernatant, the immobilization was stopped
by filtration through POEBEL Buchner funnel with perforate plate
No. 2, then rinsed with 10 vol of 25 mM phosphate buffer pH 7 and
◦
stored at 4 C.
3
(
.2.3. Immobilization of CRL on naphthalene-agarosa
naphthyl-CRL)
First, the preparation of the naphthalene-activated agarose
naphthyl) was conducted. The procedure consisted in two steps:
(
1
[
) preparation of epoxy-activated agarose as described elsewhere
34]; and 2) naphthalene-activated epoxy-agarose. Based on a pre-
vious report [35], a suspension of 10 g of epoxy-agarose in 100 mL of
5 mM tionaphtalene in water/dimethylsulfoxide (20:80) was pre-
pared. The suspension was maintained under gentle agitation for
h at room temperature. Afterwards, the support was filtered and
rinsed with 10 vol (100 mL each time) of water/acetone (20:80).
Once naphthyl-agarose was obtained, the immobilization of CRL
on this support was carried out following the methodology above
described for octyl-sepharose.
2
. Materials
The reagents racemic 1-phenylethyl acetate (rac-1),
1
racemic 1-phenylethanol (rac-2), (R or S)-1-phenylethanol
or S-2), ethyl 2-hydroxy-4-phenylbutyrate (rac-3),
ethyl (R or S)-2-hydroxy-4-phenylbutyrate (R or S-3), 1-
ethyl-3-[3-dimethylaminopropil]carbodiimide (EDC) and
N-hydroxysuccinimide (NHS) were purchased from Pierce
Scientific (Mexico City, Mexico). Glutaraldehyde (GA) (25% in
water solution), sodium borohydride, polyethyleneimine (PEI)
molecular weight of 1300 g/mol, polyethylenglycol (PEG) molec-
ular weight of 600 g/mol, bovine serum albumin (BSA), Triton®
X-100, ethanolamine, ethylenediamine, trioctanoin, p-nitrophenyl
butyrate (pNPB) were acquired from Sigma (St. Louis, MO, USA).
All other reagents were analytical or HPLC grade. Supports as
cyanogen bromide activated agarose (CNBr) and octyl-sepharose
(
R
2
3.2.4. Immobilization of CRL on glyoxyl-agarose (glyoxyl-CRL)
Fully glyoxyl-activated agarose (glyoxyl) was prepared as
described elsewhere [36]. Afterwards, the immobilization was con-
ducted by mixing 10 mL of enzyme solution (1 mg of enzyme
powder per mL of 100 mM bicarbonate buffer pH 10 with 40%
PEG [37] and 0.05% Triton® X-100 p/v) with 1 g of glyoxyl-agarose.
4
BCL (octyl) were purchased from GE Healthcare (Uppsala,
◦
The suspension was maintained under gentle agitation at 4 C.
Sweden). Crosslinked agarose beads 6 BCL were purchased from
Agarose Beads Technologies (Madrid, Spain). The enzyme Candida
rugosa lipase (CRL) TVII (with 20 mg of protein/g of powder) was
purchased from Sigma (St. Louis, MO, USA).
The immobilization curse was followed by measuring the activity
in both the supernatant and the suspension as far as no activ-
ity was detected in the supernatant. The attachment of the 100%
of the enzyme on the support was done in 15–30 min; however,

3
4
S. Velasco-Lozano et al. / Journal of Molecular Catalysis B: Enzymatic 130 (2016) 32–39
Table 1
Carrier-bound and carrier-free immobilization of CRL.
Type
Derivative
Enzyme-support interaction
Probably enzyme orientation
Y(%)
rAe(%)
Carrier-bound
CNBr
GA
Covalent unipoint binding
1st – ionic adsorption2nd – covalent binding
Covalent multipoint binding
Ionic adsorption
Binding of amino N-terminus
1st – negatively charged area2nd – lysine abundant area
Lysine abundant area
Negatively charged area
Hydrophobic area
54
100
99
83
100
41
15
8
7
14
53
24
22
15
Glyoxyl
MANAE
Octyl
Naphthyl
c-CLEA
a-CLEA
Hydrophobic adsorption
Carrier-free
none
none
Aspartic and glutamic acids abundant area
Lysine abundant area
55
81
In all cases the activity was measured with pNPB as substrate.
Immobilization yield (Y) = (initial activity – activity in the supernatant)/initial activity.
Relative expressed activity (rAe) = [final expressed activity of the immobilized enzyme/(initial activity – activity in the supernatant)].
◦
the immobilization was maintained in curse through 24 h at 4 C
in order to promote the formation of multiple enzyme-support
bonds. Subsequently, a reduction was done by addition of sodium
borohydride (1 mg/mL of suspension) followed by soft agitation for
acetonitrile) in 2 mL of 25 mM phosphate buffer pH 7 in a magnetic
stirred cell at 25 C. The released p-nitrophenol was monitored at
◦
348 nm during 5 min of reaction. Calibration curve of p-nitrophenol
in the same conditions was carried out. One unit of lipase activity
was defined as the amount of enzyme required for release 1 ␮mol
of p-nitrophenol per minute.
3
0 min at room temperature. Once the reduction was finished, the
immobilized derivative was abundantly washed with water and
was equilibrated for 5 min with 10 mL of 25 mM phosphate buffer
◦
pH 7, filtered and stored at 4 C.
3.5. Enzymatic hydrolysis of racemic 1-phenylethyl acetate
(
rac-1)
3.2.5. Immobilization of CRL on MANAE-agarose (MANAE-CRL)
Monoamino-N-aminoethyl activated agarose (MANAE-agarose)
The hydrolysis of rac-1 was carried out by adding CLEA
300–400 ␮g of protein) or immobilized derivative (500 mg) to 3 mL
of 5 mM of rac-1 in buffered solution (10 mM of Tris or sodium
was prepared as previously reported [38]. The immobilization was
done by mixing 10 mL of enzyme solution (1 mg of enzyme powder
(
®
per mL of 25 mM phosphate buffer pH 7 and Triton X-100 0.05%
acetate, for pH 7 or pH 5, respectively). The reaction mixture was
w/v) with one gram of the support. The suspension was maintained
◦
maintained under gentle agitation (at 25 or 4 C, as required), up to
◦
under gentle agitation at 4 C until no activity was present in the
attain 10–15% of hydrolysis degree of the substrate. For the hydrol-
ysis degree determination, samples of the reaction mixture were
withdrawn and analyzed in an HPLC (Spectra System P4000) cou-
pled to an UV-diode array detector (Spectra System SN4000) and
eluted with a Kromasil C18 (25 cm × 0.4 cm) column. The elution
of compounds was conducted with a mobile phase of ammonium
phosphate buffer (10 mM and pH 7.0) in acetonitrile/water (35:65,
v/v), with an isocratic flow of 1.0 mL/min. The compounds were
detected at 205 nm. Retention times for rac-2 and rac-1 were 4.6
and 22.0 min, respectively. One unit of activity was defined as the
amount of enzyme needed for release one ␮mol of (R) or (S)-2
per minute. The conversion degree was estimated after relation-
ship of the peak’s area and a calibration curve in the same elution
conditions.
supernatant (around 30 min). The immobilization was stopped by
filtration with POEBL Buchner funnel with perforate plate No. 2,
and rinsed with 10 vol of 25 mM phosphate buffer pH 7 and stored
◦
at 4 C.
3
.2.6. Immobilization of CRL on glutaraldehyde-agarose (GA-CRL)
Glutaraldehyde-activated agarose (GA-agarose) was prepared
as described elsewhere [39]. The immobilization of CRL on GA-
agarose was carried out according a described methodology [40].
Ten millilitres of enzyme solution (1 mg of enzyme powder per mL
of 25 mM phosphate buffer pH 7 with 0.05% w/v Triton® X-100)
were mixed with one gram of fresh GA-agarose. The suspension
◦
was maintained under gentle agitation at 4 C until no activity was
detected in the supernatant (around 30 min). The immobilization
was stopped by filtration in a POBEL Buchner funnel with perfo-
rate plate No. 2, rinsed with 10 vol of 25 mM phosphate buffer pH
The enantiomeric excess (ee) of released (S)-2 was determined
by reverse chiral HPLC (Spectra System P4000) coupled to an UV-
diode array detector (Spectra System SN4000) with a Chiralcel
OD-R (250 mm × 4.6 mm) column. To assure a first-order enzyme
kinetic, samples with 10–15% of substrate’s hydrolysis degree were
employed. The separation of 2 enantiomers was done with an iso-
cratic mobile phase of ammonium phosphate buffer (10 mM at
pH 7.0) in an acetonitrile/water mixture (35:65% v/v), with a flow
of 0.5 mL/min. The enantiomers were detected at 205 nm, with a
retention times of 13.5 and 14.7 min for (S)- and (R)-2, respec-
tively. Enantioselectivity (E) was calculated with the Chen equation
[42].
7
in order to remove remaining glutaraldehyde and detergent, and
◦
stored at 4 C.
3.3. Polymer coating of immobilized CRL (PEI-coating)
Based on a reported methodology [41], a suspension of CLEA
(
30–40 ␮g of protein/mL) or 1 g of support-immobilized CRL with
1
6 mL of PEI (25 kDa) solution (25 mg/mL adjusted at pH 7) was
◦
maintained at soft agitation at 4 C for 16 h. Afterwards, the PEI-
coated derivatives were filter and washed with abundant 25 mM
phosphate buffer pH 7. The physically modified derivatives were
3.6. Enzymatic hydrolysis of racemic ethyl 2-hydroxy-4-
◦
stored at 4 C.
phenylbutyrate (rac-3)
3
.4. Activity assay
The enzymatic hydrolysis of rac-3 was carried out according
to a previous report [43]. A suspension of CLEA (300–400 ␮g of
protein) or immobilized derivative (500 mg) in 3 mL of 5 mM of
rac-3 in 5 mM of buffered solution (10 mM of sodium acetate or
sodium phosphate, for pH 7 or pH 5, respectively) was prepared.
The reaction mixture was maintained under gentle agitation (at 25
Lipase activity during immobilization procedures was deter-
mined by the hydrolysis of p-NPB as substrate. To start the reaction,
0–100 ␮L of enzyme solution or suspension properly diluted were
added to a reaction mixture containing 20 ␮L of pNPB (50 mM in
5

S. Velasco-Lozano et al. / Journal of Molecular Catalysis B: Enzymatic 130 (2016) 32–39
35
◦
or 4 C) until the achievement of 10–15% of substrate hydrolysis
soluble enzyme. On the other hand, CRL covalently immobilized
on agarose activated with aldehyde groups -glutarladehyde (GA)
and glyoxyl-carriers- under alkaline conditions gave rise to inac-
tive enzymes. These types of immobilization chemistry enhance
enzyme stability towards temperature, pH, and solvents, among
others [39,40,44,51]. However, immobilization conditions (pH 10
and sodium borohydride) on carriers activated with aldehyde
groups are deleterious for several enzymes [52]. To address such
inactivation issue during the immobilization, CRL was immobi-
lized on glyoxyl-agarose in the presence of 40% PEG allowing
the stabilization of the enzyme under alkaline conditions [37].
Under such conditions, 99% of CRL was immobilized express-
ing only 7% specific activity of the soluble enzyme. Alternatively,
CRL was immobilized on GA-agarose under neutral pH condi-
tions. In this immobilization chemistry, the negatively charged
enzyme regions are firstly adsorbed to positively charged surface
of the carrier, this ionic immobilization is followed by the cova-
lent binding between GA groups and lysine residues located at
the carrier and enzyme surface. In spite of the mild immobiliza-
tion conditions, CRL immobilized on GA-agarose only expressed
8% specific activity of the soluble enzyme although the immobiliza-
tion yield was 100% (Table 1). Likewise, reversible immobilization
of CRL on agarose activated with primary amine groups (MANAE-
agarose) was carried out under soft conditions (neutral pH, low
ionic strength and short immobilization times). This immobiliza-
tion chemistry orients CRL through its most basic regions (rich in
Asp and Glu). On this aminated support, 83% of CRL was immo-
bilized expressing 14% of the enzyme specific activity in solution.
When we carried out the same immobilization protocol with lower
enzyme loads (ten times less), 83% of CRL was immobilized but
the specific activity of the immobilized enzyme was nearly 42%
of its soluble counterpart. This result points out that the activ-
ity loss after the immobilization process is mainly due to transfer
limitations rather than an intrinsic inactivation caused by the
enzyme-carrier interaction. Beyond the ionic interaction, CRL was
also reversibly immobilized through hydrophobic forces. In this
regard, the enzyme was immobilized on agarose activated with
either octyl (octyl-sepharose) or naphtyl (naphtyl-agarose) groups.
This immobilization chemistries orient CRL through its hydropho-
bic active site by hydrophobic interaction between hydrophobic
residues on the vicinity of the catalytic pocket and the either alkyl
chain or aromatic rings on the carrier surface, resembling an oil-
water interface at low ionic strength [53]. Adsorption of CRL on
both octyl- and napthyl-agarose resulted in the highest expressed
specific activity among all the immobilized preparations herein
studied (Table 1). Besides both derivatives presented the higher
expressed specific activity of the enzyme, octyl-sepharose immo-
bilized CRL was much more active than napthyl-agarose. In conrast,
100% of CRL was immobilized on octyl-sepharose while only 41%
of CRL could be immobilized on napthyl-agarose. These differences
might be attributed to a higher affinity of CRL for alkyl chains than
for aromatic rings. This fact may be explained because a better geo-
metric congruence between the CRL active site and the octyl chains,
since CRL exhibits a long, narrow hydrophobic tunnel which accom-
modates the acyl moiety of the substrate [54]. Alternatively, CRL
was aggregated and covalently cross-linked by using two different
cross-linking chemistries 1) using glutaraldehyde as cross-linking
agent that covalently bridge inter- or intraprotein lysine residues
(a-CLEA) or 2) using carbodiimide as carboxy-activating agent fol-
lowed by polyethyleneimine cross-linking that covalently bridges
intra- or intermolecular bridges between two carboxylic groups
from either Asp or Glu residues (c-CLEA). Cross-linking with glu-
taraldehyde irreversibly immobilized a higher yield of CRL than
cross-linking with carbodiimide. However, both CLEA showed sim-
ilar CRL specific activities; around 20% of the soluble enzyme.
Carrier-free immobilized derivatives of CRL drove to 2-fold more
degree. Hydrolysis degree was determined by HPLC (Spectra Sys-
tem P4000) coupled to an UV-diode array detector (Spectra System
SN4000) with a Kromasil C18 (25 cm × 0.4 cm) reverse phase col-
umn. The elution of compounds was done with an isocratic mobile
phase of ammonium acetate buffered solution (10 mM and pH 2.3)
in an acetonitrile/water mixture (40:60 v/v) and a constant flow of
1
.0 mL/min. Analytes were detected at 225 nm with retention times
of 3.2 and 10.2 min for the product, racemic hydroxy-phenylbutyric
acid (rac-4) and the substrate (rac-3), respectively. One unit of activ-
ity was defined as the amount of enzyme needed for release one
mol of (R)- or (S)-4 per minute. The conversion degree was esti-
mated after relationship of the peak’s area and a calibration curve
in the same elution conditions.
␮
The ee of released (R)-3 was determined by reverse chiral HPLC
Spectra System P4000) coupled with an UV-diode array detector
(
(
Spectra System SN4000) and a Chiralcel OD-R (250 mm × 4.6 mm)
column. Samples with 10–15% of substrate’s hydrolysis degree
were analyzed in order to ensure a first-order enzyme kinetic. HPBE
enantiomers were eluted with an isocratic mobile phase of ammo-
nium phosphate buffer (10 mM at pH 2.3) in an acetonitrile/water
mixture (20:80, v/v) and a flow of 0.5 mL/min. Enantiomers were
detected at 225 nm with retention times of 23.1 and 25.3 min for
(
S)- and (R)-3, respectively. Enantioselectivity was calculated with
Chen equation [38].
4
. Results and discussion
4
.1. Immobilization of CRL by both carrier-bound and carrier-free
techniques
Immobilization is frequently associated to the loss of enzymatic
activity and the alteration of some catalytic properties. Hence, the
study of a variety of matrixes functionalized with different reactive
groups enabled to orient CRL through different enzyme regions.
Both enzyme orientation and immobilization chemistry directly
affect the catalytic properties of the immobilized biocatalyst [20].
As model carrier we used agarose beads because of its versatility to
be functionalized with many different reactive groups, its compati-
bility with biomolecules and its mechanical stability to be used in a
plethora of reactor designs [36,44]. On the other hand, the commer-
cial preparation of CRL used in this work is not pure since is mainly
composed by the isoforms Lip1 and Lip3 [45]. However, industrial
biocatalysts must meet the cost requirements regarding to their
large scale applications; hence, purified enzymes represent a very
high expensive choice for such purpose. Besides, available commer-
cial CRL preparations contain more than one isoform of this enzyme
and they have been widely applied in the successful resolution of
racemic mixtures [17,46–48] thus confirming its potential as robust
industrial biocatalyst.
In order to find a highly enantioselective insoluble CRL, we
screened six immobilization chemistries on pre-existing sup-
ports and two cross-linking chemistries on enzyme aggregates.
Table
1 shows the immobilization chemistry, the proposed
enzyme orientation and the resulting immobilization parameters
for each immobilized preparation. Among overall immobilization
chemistries, the adsorption on hydrophobic supports achieved the
highest expressed activity of CRL, while multipoint covalent ones
drives the least active biocatalysts.
CNBr-activated support immobilizes CRL through its N-terminus
establishing only one (or very few) enzyme-carrier bond. Conse-
quently, the enzyme suffers low modification degree, exhibiting
nearly the same catalytic properties as the free enzyme [49,50].
This immobilization chemistry drives the quantitative immobi-
lization of CRL expressing only 15% of the specific activity of the

3
6
S. Velasco-Lozano et al. / Journal of Molecular Catalysis B: Enzymatic 130 (2016) 32–39
Fig. 1. Molecular substrate binding models for enantiomers of rac-1 suggested by molecular modeling done through SwissDock server (http://www.swissdock.com.ch/).
CRL 3D structure was PDB 1CRL. (A) (S)-1 with ꢀG = −6.03 kcal/mol, (B) (R)-1 with ꢀG = −6.15 kcal/mol. Lighted in orange and blue the hydrophobic and the hydrophilic
enzyme zones, respectively. In black, two amino acids of the catalytic triad (serine 209 and histidine 449). Models A and B represent the lowest energy binding models for
each enantiomers. Data and adjustment visualization were obtained with UCSF Chimera program. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
Table 2
Effect of temperature and co-solvent addition in the hydrolysis of rac-1 catalyzed by CRL derivatives.
◦
◦
◦
4 C, pH 7, acetonitrile (30%)
Biocatalysts
25 C, pH 7
4
C, pH 7
Specific activity (U/g)1
E (S)2
Specific activity (U/g)1
E (S)2
Specific activity (U/g)1
0.4d
E (S)2
c
6a
d
a,b
41e
CNBr
GA
277
56
5
7
d
b
a
a
a,b
b
2
1
6
0.1
19
e
a
a,b
a
b
c
glyoxyl
octyl
naphthyl
MANAE
c-CLEA
a-CLEA
176
6
2
6
0.1
27
f
a
e
c
c
d
752
7
19
10
0.3
34
g
a
b,c
b,c
b
f
45
6
3
8
0.1
106
h
b
c,d
a
e
g
602
10
4
5
0.5
>200
a
a
a,b
a
b
a
161
5
2
6
0.1
14
b
a
a,b
a
a
a
20
6
2
5
0.05
13
Reaction conditions were 5 mM of rac-1 in buffer Tris (10 mM, pH 7). a,b,c,d,e,f,gMean values with different letters are statistically different (P < 0.05). Values represent the
average of two independent assays.
1
Specific activity was expressed as units per gram of immobilized protein.
E was calculated with Chen equation.
2
active immobilized enzymes than their multipoint covalent carrier-
bound counterparts, demonstrating lower inactivating effects on
CLR’s activity.
presented the lowest activity. Besides, more acid pH decreased
the lipase activity towards rac-1, but the activity trend for each
immobilized preparation was kept regarding to pH 7, being the
CRL immobilized on both MANAE-agarose and octyl sepharose the
most active ones. This effect is supported by the optimum activity
of CRL, which was reported at pH 6.5, maintaining 80% of its activ-
ity at pH 7, whilst at pH 6 only the 55% was retained [55]. Under
acidic conditions, CRL presented a poor selectivity towards the S-
enantiomer as well as under pH 7. In the tested enantioselective
resolution, CRL was not positive affected by the pH of the media,
unlike its enhanced enantioselectivity in the resolution of mandelic
acid esters at acidic pH conditions [18].
The difference of the immobilization parameters among the
different CRL immobilized preparation suggests that the enzy-
matic properties of this lipase may be quite different from one
preparation to others. Therefore, here we present a library of CRL
heterogeneous catalysts with different orientation and immobiliza-
tion chemistries that may alter the CRL performance in different
biotransformations. This library will be the starting point to screen
for high lipase enantioselectivity towards two different racemic
esters.
The effect of temperature on both activity and enantoselectiv-
ity was evaluated at pH 7 with and without addition of acetonitrile
4
.2. Enantioselective hydrolysis of racemic 1-phenylethyl acetate
(
Table 2). Although, for all the immobilized biocatalysts, the activity
(
rac-1)
◦
is strongly reduced (10–60 times) at 4 C, the enantioselectivity at
4
◦
◦
C and 25 C is similar. The enantioselectivity was not improved
We used eight immobilized CRL preparations to hydrolyze rac-1
by reducing the reaction temperature as it has been observed for
this enzyme in the resolution of l-lactic acid [56] and in the ester-
ification of 1-phenylethanol with caproic acid [57] and also for
other enzymes [9,43]. In order to improve the enantioselectivity,
acetonitrile was added to the reaction media as co-solvent and
in order to produce the corresponding enantiomer of the sec-
ondary alcohol (Scheme 1A). Firstly, this biotransformation was
carried out in aqueous media at two different pH values. Gen-
erally, all CRL derivatives presented very low selectivity towards
the S enantiomer (Supporting information Table 1S). However, the
activity strongly varied among the immobilized biocatalysts, being
the CRL immobilized on MANAE-agarose and octyl-sepharose the
most active preparations, while the a-CLEA and naphtyl-derivative
◦
the reaction was carried out at 4 C. As expected, the presence
of acetonitrile in the reaction media decreased the enzyme activ-
ity by 1–2 orders of magnitude, mainly because of the enzyme

S. Velasco-Lozano et al. / Journal of Molecular Catalysis B: Enzymatic 130 (2016) 32–39
37
Fig. 2. Molecular substrate binding models for enantiomers of rac-3 suggested by molecular modeling done through SwissDock server (http://www.swissdock.com.ch/). CRL
D structure was PDB 1CRL. Models A and B represent the lowest energy binding models for each enantiomers. Models C and D represent the lowest energy binding models
3
for each enantiomers in which the substrate’s ring is oriented towards the hydrophobic tunnel. A) (S)-3 with ꢀG = −6.26 kcal/mol, B) (R)-3 with ꢀG = −6.23 kcal/mol, C) (S)-3
with ꢀG = −5.58 kcal/mol and D) (R)-3 with ꢀG = −5.57 kcal/mol. Lighted in orange and blue the hydrophobic and the hydrophilic enzyme zones, respectively. In black, two
amino acids of the catalytic triad (serine 209 and histidine 449). Data and adjustment visualization were obtained with UCSF Chimera program. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 3
Effect of co-solvent addition in the hydrolysis of rac-3 catalyzed by CRL derivatives.
◦
◦
C, acetonitrile (30%)
Biocatalysts
4
C
4
Specificactivity(U/g)1
87f
E (S)2
Specific activity (U/g)1
2.0c
E (S)2
a,b,c
12b,c
CNBr
GA
8
d
b,c
d
c,d
35
9
3.1
14
c
c
a
b
glyoxyl
octyl
naphthyl
MANAE
c-CLEA
a-CLEA
16
11
5
8
9
0.4
9
f
a
e
a
87
4.3
0.9
4
1
b
a,b,c
b
a
7
e
b,c
b
a
49
1.1
1.3
3
b
c
b,c
e
9.5
10
39
a
a,b
b
d
1.5
6
1.1
18
Reaction conditions were 5 mM of rac-3 in acetate buffer (10 mM, pH 5) at 4 C. a,b,c,d,e,fMean values with different letters are statistically different (P < 0.05). Values represent
◦
the average of two independent assays.
1
Specific activity was expressed as units per gram of immobilized protein.
E was calculated with Chen equation.
2
inactivation/inhibition triggered by the acetonitrile molecules [58].
However, under such conditions enantioselectivity of CRL was
highly increased in all cases. The organic solvent presence is an
important factor which might modify the enzyme’s enantioselec-
tivity. However, a general correlation between the physiochemical
properties of the organic solvent (log P value or dielectric constant)
and a higher enantioselectivity was not found [10]. Noteworthy,
the highest enantioselective (E > 200) was obtained with the CRL
immobilized on MANE-agarose. We consider that the active site of
this immobilized biocatalyst is re-shaped to more selectively bind
the S-enantiomer of 1 by the cooperative effect of the CRL orienta-
tion through its most basic regions and the new solvatation sphere
of the active site promoted by the acetonitrile molecules. Therefore,
this artificial molecular environment seems to be only stable under
low temperature conditions because addition of acetonitrile to the
reaction media at 25 C had a deleterious effect on both activity and
◦
enantioselectivity (results not shown). Probably, that temperatures
◦
above 4 C may alter the optimal local conformation of CRL due to
some negative molecular vibrations thermally triggered.
With the aim of understanding the molecular binding of rac-1 to
the active site, we docked this substrate into the CRL active site (PDB
1CRL) by the UCSF Chimera program [59]. We observed a different
orientation of the aromatic ring for each enantiomer (Fig. 1A and B).
In the case of (S)-1, the aromatic ring is aligned to the hydrophobic
tunnel locating the carbonyl group very close to the catalytic ser-
ine (S209). Contrarily, the aromatic ring of the (R)-1 is stuck into

3
8
S. Velasco-Lozano et al. / Journal of Molecular Catalysis B: Enzymatic 130 (2016) 32–39
one hydrophobic cavity, resulting in a still possible but less catalyt-
ically productive conformation (Fig. 1B). These slight differences in
the substrate binding may explain the slight enantiopreference of
CRL towards S-enantiomer. Thereby, a little change in the molec-
ular vicinity of the catalytic pocket caused by one specific enzyme
orientation, as well as substrate solubility differences promoted by
the acetonitrile, might build a more S-preferential active site as was
experimentally observed.
the c-CLEA preparation. Another insight that reinforces the neg-
ligible role of PEI is that, a-CLEA showed lower enantioselectivity
than c-CLEA even though both were prepared in the presence of
PEI. Hence, we suggest that such enantioselectivity increase might
be due to conformational changes of the catalytic pocket of the
enzyme, as a result of the merged effect of carboxyl-cross-linking of
the enzyme and the reaction conditions. For a better understanding,
we obtained the molecular binding models of the catalytic pocket of
CRL and the enantiomers of rac-3 with different orientations of the
substrate’s ring [facing outward (Fig. 2A and B) or inward (Fig. 2C
and D) of the hydrophobic tunnel]. Such modeling revealed that
4.3. Enantioselective hydrolysis of racemic ethyl
2-hydroxy-4-phenylbutyrate (rac-3)
(S)-3 is equally favorable as (R)-3 since both present almost the
Besides alcohols, enantiopure carboxylic acids constitute a wide
same formation energies in the different fitting models, which is in
agreement with the low observed E values. Therefore, the enhanced
enantioselectivity of c-CLEA, might be more related to the resulted
enzyme 3D configuration after its immobilization in combination
with the reaction medium conditions, and not for a different orien-
tation of the substrate’s moieties within the enzyme’s hydrophobic
pocket.
field of potential application of enantioselective lipases. Thereby, in
this study we also evaluated the aqueous hydrolysis of rac-3 cat-
alyzed by the eight lipase immobilized preparations (Scheme 1B).
Initially, the effect of pH on the biocatalysts’s enantioselectivity
and activity was evaluated. All biocatalysts showed major prefer-
ence for the hydrolysis of the (S)-enantiomer at the two tested pH
values (Table 2S). However, observed E values were relatively low
(
Emax pH 7 < 4 and Emax pH 5 < 9). Regarding the activity, five bio-
5. Conclusions
catalysts showed higher activity at pH 7 than at pH 5. As in the
resolution of (S)-2, this effect might be attributed to the major
activity that CRL exhibits at pH 7. Nonetheless, the activity of the
MANAE-CRL (aminated support) was higher at pH 5 than at pH 7.
The latter results might be related to a weak enzyme-support inter-
action, since in this support most of the salt bridges formed by the
enzyme-carboxyl groups and the support-amino groups would be
cut off at pH 5. Therefore, the immobilized enzyme on this aminated
support at pH 5 expresses more activity due a less rigidification
effect.
Despite most biocatalysts presented higher activity at pH 7 than
at pH 5, all the immobilized preparations were more enantioselec-
tive at pH 5; however, in all cases the obtained E values were too
low for practical utilization.
In order to improve the enantioselectivity of the immobilized
biocatalysts, temperature and co-solvent addition were evaluated
We have constructed a library of different carrier-bound and
carrier-free insoluble preparation of CRL. By using a combinato-
rial approach that involved immobilization techniques and reaction
engineering we have been able to screen 8 different immobi-
lized CRL variants in 4 different reaction conditions, controlling
parameters such as temperature, pH and media polarity. We have
found different heterogeneous biocatalysts able to enantioselec-
tively hydrolyze the S-enantiomer of two different esters with high
E values. The immobilization of CRL on MANAE agarose hydrolyzed
rac-1 with the highest rate and highest enantioselectivity (E > 200)
◦
under pH 7, 4 C and 30% (v/v) acetonitrile. Likewise, aggregated
CRL and cross-linked with cardodiimide hydrolyzed rac-3 with the
◦
highest enantioselectivity (E = 39) under pH 5, 4 C and 30% (v/v)
of acetonitrile. Therefore, low temperatures and presence of co-
solvents enhance the CRL enationselectivity towards the S-isomer,
while pH had negligible effect for the hydrolysis of rac- 1 but noto-
rious for the hydrolysis of rac-3. Finally, in this work we have
demonstrated the importance of the immobilization protocol in
the final catalytic properties of the heterogeneous biocatalyst as
result of the variety of orientations and structural conformation
of the immobilized enzyme. Moreover, combinatorial prepara-
tion of heterogeneous biocatalysts can efficiently merge to other
strategies as reaction medium engineering to discover highly enan-
tioselective heterogeneous biocatalysts. Nowadays, several works
have demonstrated the effectiveness of carrier-bound immobiliza-
tion techniques as suitable and feasible tool for the enhancement
and modulation of enantioselectivity. However, herein we demon-
strated that carrier-free immobilization is also available for the
same purpose, with the advantage that is a less expensive choice.
Thereby, this work contributes to the knowledge and understand-
ing of lipase enantioselectivity behaviour.
(
Table 3). All biocatalysts showed lower activity (around 4-times
◦
less) when temperature was decreased to 4 C. However, at this
lower temperature enantioselectivities were 1.2-times higher.
Then, we added acetonitrile to the reaction medium, which
promoted the decreasing of the enzymatic activity for all the immo-
bilized preparations; likewise it occurred for rac-1. The variation
of the enantioselectivity was unpredictable since glyoxyl-, octyl-,
naphthyl- and MANAE-CRL derivatives presented lower enantiose-
lectivity in the presence of acetonitrile than in full aqueous media.
Conversely, CNBr-, GA-CRL and both CLEA preparations were more
enantioselective in presence of acetonitrile, achieving the highest
enantioselectivity value of this study with the c-CLEA (E = 39). The
improved enantioselectivity of c-CLEA might be related to the com-
bination of tree effects: 1) conformational changes of the catalytic
site, as a result of the enzyme-enzyme interactions, 2) the reaction
◦
medium conditions (4 C and 30% acetonitrile); and 3) the posi-
tive charged microenvironment in the surroundings of the enzyme,
which was provided by the PEI.
Declaration of interest
To explain the positive effect of PEI on the enantioselectiv-
ity of CRL, we coated with PEI all the derivatives where CRL was
immobilized on pre-existing carriers and tested them for the stere-
oselective hydrolysis of (S)-3. This physical modification provides
an increase of hydrophylicity and charge microenvironment of the
enzyme. The latter modification was not performed in CLEA since
these derivatives are prepared in the presence of PEI. Immobilized
preparation coated with PEI- showed negligible variations in enan-
tioselectivity; E values were indeed lower when PEI was coating
the enzyme (data not shown). Therefore, we discard the effect of
PEI as major contributor to enhance the CRL enantioselctivity in
The authors declare no competing financial interest.
Acknowledgements
This work was supported by the Mexican Council for Science
and Technology (CONACyT) by the project No. 154004. S. Velasco
is grateful for the scholarship received from CONACYT. We would
like to thank IKERBASQUE, Basque foundation for Science for the
funding to PhD Fernando López Gallego.

S. Velasco-Lozano et al. / Journal of Molecular Catalysis B: Enzymatic 130 (2016) 32–39
39
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