Highly enantioselective biocatalysts by coating immobilized lipases with polyethyleneimine

Article abstract of DOI:10.1016/j.catcom.2010.04.010

Polyethyleneimine (PEI) modification on immobilized lipases greatly enhanced their enantioselectivity in the kinetic resolution of (±)-2-hydroxy-phenylacetic acid methyl ester. The enantiomeric ratio (E) of CNBragarose-CRL rose from 8 (without coating) to 20 (ee=90%) after PEI coating in the hydrolysis at pH 5. In the case of CAL-B, the coating highly improved the enantioselectivity of the immobilized lipase from E=1.5 (without coating) to E>100 (ee>99%). Moreover, this coating strategy improved the stability of the biocatalyst at high temperatures and in the presence of high co-solvent concentrations.

Full text of DOI:10.1016/j.catcom.2010.04.010

Catalysis Communications 11 (2010) 964–967
Contents lists available at ScienceDirect
Catalysis Communications
journal homepage: www.elsevier.com/locate/catcom
Highly enantioselective biocatalysts by coating immobilized lipases
with polyethyleneimine
1
2
Zaida Cabrera , Melissa L.E. Gutarra , Jose M. Guisan ⁎, Jose M. Palomo ⁎
Departamento de Biocatálisis, Instituto de Catálisis (CSIC), Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 March 2010
Received in revised form 9 April 2010
Accepted 14 April 2010
Available online 24 April 2010
Polyethyleneimine (PEI) modification on immobilized lipases greatly enhanced their enantioselectivity in
the kinetic resolution of (± )ꢀ2ꢀhydroꢁyꢀphenylacetic acid methyl ester. The enantiomeric ratio (E) of CNBrꢀ
agaroseꢀCRL rose from 8 (without coating) to 20 (ee=90%) after PEI coating in the hydrolysis at pH 5. In the
case of CALꢀB, the coating highly improved the enantioselectivity of the immobilized lipase from E=1.5
(without coating) to EN100 (eeN99%). Moreover, this coating strategy improved the stability of the
biocatalyst at high temperatures and in the presence of high coꢀsolvent concentrations.
Keywords:
Protein modification
Polymers
©
2010 Elsevier B.V. All rights reserved.
Enantioselectivity
Candida antarctica lipase
Candida rugosa lipase
1
. Introduction
strongly adsorb many different proteins at neutral pH value [14] and
stabilize multimeric enzymes by preventing subunit dissociation [15].
Furthermore, the PEIꢀcoating strategy was used to stabilize
immobilized lipases in anhydrous organic solvents [16], or immobiꢀ
lized penicillin G acylase in high coꢀsolvent aqueous media [17].
This coating of all negative areas on the protein surface could
introduce a hydrophilic environment around the active site and
perhaps modify the electrostatic interactions and therefore the shape
of the active site (Fig. 1).
Herein an efficient methodology to greatly improve the enantiosꢀ
electivity of lipase from Candida antarctica B (CALꢀB) and Candida
rugose (CRL) has been developed by coating the enzymes in
immobilized form with polyethyleneimine (Fig. 1). The kinetic
resolution of (± )ꢀ2ꢀhydroꢁyꢀphenylacetic acid methyl ester 1, a
particular interesting building block in the synthesis of different drugs
[18–20] was used as model reaction.
The development of new biocatalysts with high selectivity, activity
and stability for the application on different kinds of chemical
reactions is of great interest for the implementation of enzymes on
industrial processes.
Many different strategies have been developed to improve the
catalytic properties of enzymes, such as rational protein engineering,
directed evolution [1–3], immobilization methods [4,5] or chemical
modification of specific amino acids on the protein [6].
Particularly, the immobilization approach was a very efficient and
interesting strategy in tuning the catalytic properties of lipases [7], in
part because of the peculiar catalytic mechanism of these enzymes [8].
Also some chemical modifications using polyethylene glycol or
activated deꢁtrans have given good results [9,10].
Polyethylenimine (PEI) is a polymer with high density of ionized
tertiary, secondary, and primary amino groups. This fact may permit
the strong ionic eꢁchange of the polymer on any area of a protein
surface containing anionic groups. Thus, this polymer has been used
to stabilize proteins in solution by preventing oꢁidation, aggregation,
and so on [11–13]. Also, supports coated with PEI were used to
2. Materials and methods
2.1. Materials
C. antarctica lipase (fraction B) (CALꢀB) was purchased from
Novozymes. C. rugose lipase, high molecular weight branched PEI
(
HMwPEI, Mw=25,000, PDI=2.5, molar ratio of primary/secondary/
⁎
Corresponding authors. C/Marie Curie 2, Cantoblanco Campus UAM, 28049, Madrid,
Spain. Tel.: +34 91 585 4809/5478; faꢁ: +34 91 5854760.
tertiary amine=approꢁ. 1/1.2/0.76), pꢀnitrophenylbutyrate (pNPB)
and (± )ꢀαꢀhydroꢁyꢀphenylacetic acid methyl ester [(± )ꢀ1] were
purchased from Sigma Chem Co. Lipases were purified and immobiꢀ
lized on octylꢀagarose, glyoꢁylꢀagarose and CNBrꢀagarose as previꢀ
ously described [21,22]. Biocatalysts were prepared with a loading of
1 mg lipase per gram support.
E-mail addresses: jmguisan@icp.csic.es (J.M. Guisan), josempalomo@icp.csic.es
(
J.M. Palomo).
1
Permanent address: Escuela de Ingeniería Bioquímica, Pontificia Universidad
Católica de Valparaíso,Valparaíso, Chile.
2
Permanent address: Instituto de Química, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, Brazil.
1566ꢀ7367/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.catcom.2010.04.010

Z. Cabrera et al. / Catalysis Communications 11 (2010) 964–967
965
2.5. Polymer coating of immobilized enzymes
1
g of polyethyleneimine was dissolved in 20 mL of 25 mM sodium
phosphate adjusted to pH 8.0. Then, 1 g of immobilized lipase
preparation (1 mg lipase/g support) was added to this solution. The
suspension was gently stirred for 1 h and the modified enzyme
preparations were washed with distilled water (10 × 50 mL)
(
Scheme 1). The immobilized preparations were filtered under
vacuum and stored at 4 °C.
Fig. 1. PEI coating of immobilized lipases.
2.6. Enzymatic hydrolysis of (± )-1
The hydrolysis of 1 was performed by adding 0.04 g of catalyst to
1
0 mL of 5 mM substrate in 10 mM buffer solution (sodium acetate at
2
.2. Enzymatic activity assay
The activities of the soluble lipase, supernatant and enzyme
pH 5 or sodium phosphate at pH 7) at 25 °C. During the reaction, the
pH value was maintained constant by automatic titration using a
pHꢀstat Mettler Toledo DL50 graphic. The degree of hydrolysis was
analyzed by reverseꢀphase HPLC (Spectra Physic SP 100 coupled to an
UV detector Spectra Physic SP 8450) on a Kromasil C18 column
(15×0.4 cm) supplied by Analisis Vinicos (Spain). At least, triplicates
of each assay were made. The elution was performed with a mobile
phase of acetonitrile (30%, v/v) and 10 mM ammonium phosphate
(70%, v/v) at pH 2.95. The flow rate was 1 mL/min. The elution was
monitored by recording the absorbance at 254 nm.
suspension were analyzed spectrophotometrically measuring the
increment in absorbance at 348 nm produced by the release of pꢀ
nitrophenol (pNP) (∈=5.150 M
0
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 μmol of hydrolyzed pNPB per minute
per mg of enzyme (IU) under the conditions described above.
−
1
− 1
cm ) in the hydrolysis of
.4 mM pNPB in 25 mM sodium phosphate at pH 7 and 25 °C. To
2.7. Determination of enantiomeric excess
2
.3. Purification of CAL-B
The enantiomeric eꢁcess (ee) of the produced acid (2) was
analyzed by Chiral Reverse Phase HPLC. The column was a Chiracel
ODꢀR and the mobile phase was an isocratic solution of (5%, v/v)
The enzyme was purified from commercial crude eꢁtract by
interfacial adsorption as previously described [21]. 1 mL of commerꢀ
cial solution (12 mg protein) was added to 19 mL of 5 mM phosphate
buffer pH 7 and 1 g of octylꢀagarose was added and the reaction was
maintained for 3 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. This was used as
immobilized preparation for selectivity studies.
4 4
acetonitrile and (95%, v/v) 0.5 M NaClO /HClO at pH 2.3 and the
analyses were performed at a flow of 0.5 ml/min by recording the
absorbance at 225 nm.
2.8. Calculation of E value
The enantiomeric ratio (E) was defined as the ratio between the
percentage of hydrolyzed R and S isomers (from racemic miꢁture) at
hydrolysis degrees between 10 and 20%, where the reaction kinetic is
in first order. Rꢀ and S isomers were used as standard enantiomerically
pure products. Also the E value was calculated from the enantiomeric
For the preparation of the covalent immobilized catalysts, the
lipase was desorbed from the support adding 10 mL of a solution of
2
5 mM phosphate buffer pH 7 with 1% Triton Xꢀ100 (v/v) to 1 g of
octylꢀCALꢀB. SDSꢀPAGE gel of this preparation reveals just one protein
band. A solution of 0.12 mg lipase purified/mL was obtained.
p
eꢁcess of the release acid (ee ) and the conversion degree (c) using
the equation E=ln[1−c(1+ee
p
p
)]/ln[1−c(1−ee )] described by
Chen et al. [22].
2
.4. Covalent immobilization of CAL-B
2.9. Inactivation of CNBr-CAL-B preparations against T and co-solvent
A solution of 0.12 mg/mL of 25 mM phosphate buffer pH 7 with 1%
Triton Xꢀ100 of purified lipase was used in each case. The
immobilization was followed by the enzymatic assay described
previously.
Immobilization on CNBr-activated agarose: The immobilization of
CALꢀB on CNBrꢀactivated support was performed for 15 min at 4 °C to
reduce the possibilities of getting a multipoint covalent attachment
between the enzyme and the support. 10 mL of lipase solution was
added to 1 g of support for 1 h. The enzymeꢀsupport immobilization
was ended by incubating the support with 1 M ethanolamine at pH
0.5 g of biocatalyst was dissolved in 5 mL of 25 mM sodium
phosphate buffer (with 0% or 60% (v/v) acetonitrile), incubated at
25 °C or 50 °C. The remaining activity at different times was measured
by the assay described above using pNPB as substrate.
8
for 2 h. Finally, the immobilized preparation was washed with
abundant water, to eliminate the detergent. The immobilization yield
was N95%.
Immobilization on glyoxyl-agarose: The pH of lipase solution
(
10 mL) was adjusted at pH 10.1 and the solution was added to 1 g
of support and the reaction was maintained for 24 h. When the
immobilization was finished — analyzed by activity assay described
before — 20 mg of NaBH was added and after 30 min the immobilized
4
preparation was abundantly washed with distilled water. The
immobilization yield was N95%.
Scheme 1. Kinetic resolution of (± )ꢀ1 by different lipase immobilized preparations.

966
Z. Cabrera et al. / Catalysis Communications 11 (2010) 964–967
Table 1
Effect of the PEIꢀcoated on CALꢀB preparations in the hydrolysis of (± )ꢀ1 at pH 7 and
25 °C.
Immobilized preparation
Activitya
cb (%)
eec (%)
E ratio
CNBrꢀCALꢀB
1.00
1.20
0.036
0.20
5.56
12.32
20
12
14
19
20
13
70
73
88
88
61
61
6.8
7
18
19
4.2
4.2
CNBrꢀCALꢀBꢀPEI
GlyoꢁylꢀCALꢀB
GlyoꢁylꢀCALꢀBꢀPEI
OctylꢀCALꢀB
OctylꢀCALꢀBꢀPEI
a
b
c
Activity in μmol g c−a t1 min−1
c = conversion.
ee = enantiomeric eꢁcess of product (Rꢀ2).
p
.
3
. Results and discussion
The PEI modification on three different immobilized preparations
of CALꢀB was applied and the activity and enantioselectivity of these
biocatalysts were evaluated in the hydrolysis of 1 at pH 7 (Table 1,
Scheme 1).
The activity of the enzyme was improved in all cases, 5 fold for
glyoꢁylꢀCALꢀB (enzyme immobilized by multipoint covalent attachꢀ
ment), 2 fold for octylꢀCALꢀB (enzyme immobilized by hydrophobic
interactions) and 1.2 fold increment for CNBrꢀCALꢀB (enzyme
immobilized by oneꢀpoint covalent attachment). However, the
enantioselectivity of the enzyme in these eꢁperimental conditions
was not altered after coating (Table 1). The modified biocatalysts were
evaluated at pH 5 considering the effects on the lipases selectivity by
changes on the eꢁperimental conditions [23]. The activity of CNBrꢀ
CALꢀB and octylꢀCALꢀB slightly decreased after coating whereas a
huge increment in the activity value was observed for glyoꢁylꢀCALꢀB
(
44 fold) (Table 2). The enantioselectivity slightly increased at these
conditions for glyoꢁylꢀCALꢀB and octylꢀCALꢀB after coating whereas a
remarkable enhancement was observed for CALꢀB immobilized on
CNBrꢀagarose; PEI coating improved the enantiomeric ratio from 1.5
(
without coating) to more than 100 (eeN99% of Rꢀ2).
In view of this enormous effect on the enantioselectivity, the PEIꢀ
coating effect was studied on the stability of CNBrꢀCALꢀB at high
temperature or coꢀsolvent concentration (Fig. 2).
Fig. 2. Stability of PEIylation on CNBrꢀCALꢀB immobilized preparation. A. Incubation at
0 °C and pH 5. B. Incubation at 25 °C, pH 5 in the presence of 60% acetonitrile (v/v).
CNBrꢀCALꢀB (circles) and PEIꢀCNBrꢀCALꢀB (squares).
5
When the CALꢀB immobilized preparations were incubated at
5
0 °C and pH 5, the coating immobilized enzyme maintained more
than 70% activity after 24 h (Fig. 2A).
The modification of immobilized CRL with PEI caused an
improvement on the activity of 2 fold also at pH 7 and 5. However,
in the same way that with CALꢀB, the enantioselectivity was not
affected by coating when the biocatalyst was used at pH 7, although
the enantiomeric ratio (E) of the immobilized lipase rose from
8 (without coating) to 21 (ee=90% of Sꢀ2) at pH 5.
High coꢀsolvent concentration is a relevant parameter for a
possible industrial application of this strategy, especially using quite
hydrophobic substrates. The stability of these biocatalysts was studied
at pH 5 in the presence of 60% acetonitrile (Fig. 2B). The PEIꢀCNBrꢀ
CALꢀB preparation preserved more than 90% of catalytic activity after
3
0 h of incubation.
In order to eꢁpand the scope of the methodology, the effect of PEI
In conclusion, physical modifications of lipase surfaces on solidꢀ
phase by polyethyleneimine seem to be a simple and effective tool to
modify the lipase enantioselectivity. This methodology permitted to
modulate the selectivity of two known enzymes (CALꢀB and CRL)
specially immobilized on CNBrꢀagarose in the hydrolysis of (± )ꢀ1.
Thus, the CNBrꢀCALꢀB preparation after the PEI modification mainꢀ
tained the catalytic activity, improved the stability at high T and coꢀ
coating was also studied on CRL — another very useful enzyme in
biotransformations — immobilized on CNBrꢀagarose (Table 3).
Table 2
Effect of the PEIꢀcoated on CALꢀB preparations in the hydrolysis of (± )ꢀ1 at pH 5 and
25 °C.
Table 3
Effect of the PEIꢀcoated on CNBrꢀCRL preparation in the hydrolysis of (± )ꢀ1 at 25 °C and
Coating
Activitya
cb (%)
eec (%)
E ratio
10 mM phosphate buffer.
CNBrꢀCALꢀB
0.80
0.72
0.014
0.625
5.66
4.78
12
12
9
22
13
19
19
N99
85
86
79
1.5
N100
13
17
8.0
Activitya
cb (%)
eec (%)
Biocatalyst
pH
E ratio
CNBrꢀCALꢀBꢀPEI
GlyoꢁylꢀCALꢀB
GlyoꢁylꢀCALꢀBꢀPEI
OctylꢀCALꢀB
CNBrꢀCRL
CNBrꢀCRLꢀPEI
CNBrꢀCRL
7
7
5
5
17
43
10
20
22
15
15
10
68
74
75
90
6.4
7.7
8
OctylꢀCALꢀBꢀPEI
80
9.0
CNBrꢀCRLꢀPEI
21
a
Activity in μmol gcat min−1
c = conversion.
−1
a
Activity in nmol gcat min−1
c = conversion.
ee = enantiomeric eꢁcess of product (Sꢀ2).
p
−1
.
.
b
c
b
c
ee
p
= enantiomeric eꢁcess of product (Rꢀ2).

Z. Cabrera et al. / Catalysis Communications 11 (2010) 964–967
967
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Acknowledgements
This work has been sponsored by the Spanish Ministry of Science
and Innovation (Project CTQ2009ꢀ07568). We also thank the PUCV
and Programa Bicentenario de Ciencia y Tecnología (PBCT, Project
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