Enzymatic kinetic resolution of (RS)-1-(phenyl)ethanols by Burkholderia cepacia lipase immobilized on magnetic nanoparticles

Article abstract of DOI:10.1590/S0103-50532010000800019

Lipase from Burkholderia cepacia immobilized on superparamagnetic nanoparticles using adsorption and chemisorption methodologies was efficiently applied as recyclable biocatalyst in the enzymatic kinetic resolution of (RS)-1-(phenyl)ethanols via transesterification reactions. (R)-Esters and the remaining (S)-alcohols were obtained with excellent enantiomeric excess (> 99percent), which corresponds to a perfect process of enzymatic kinetic resolution (conversion 50percent, E > 200). The transesterification reactions catalysed with B. cepacia lipase immobilized by the glutaraldehyde method showed the best results in terms of reusability, preserving the enzyme activity (conversion 50percent, E > 200) for at least 8 successive cycles.

Full text of DOI:10.1590/S0103-50532010000800019

J. Braz. Chem. Soc., Vol. 21, No. 8, 1537-1542, 2010.
Printed in Brazil - ©2010 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Enzymatic Kinetic Resolution of (RS)-1-(Phenyl)ethanols by Burkholderia cepacia
Lipase Immobilized on Magnetic Nanoparticles
Lya P. Rebelo,a Caterina G. C. M. Netto,b Henrique E. Toma*,b and
Leandro H. Andrade*,a
aLaboratoryof Fine Chemistry and Biocatalysis andbSupramolecular NanotechLab, Institute of
Chemistry, University of São Paulo, 05508-900 São Paulo-SP, Brazil
A lipase proveniente da Burkholderia cepacia imobilizada em nanopartículas
superparamagnéticas usando diferentes metodologias de imobilização (adsorção e quimiosorção)
foi eficientemente aplicada como biocatalisador reciclável na resolução cinética de (RS)-1-(fenil)
etanols através de reações de transesterificação. Os (R)-ésteres e os (S)-alcoóis foram obtidos com
excelente excesso enantiomérico (> 99%), o que corresponde a um perfeito processo de resolução
cinética enzimática (conversão 50%, E > 200). As reações de transesterificação catalisadas pela
lipase de B. cepacia imobilizada pela metodologia com glutaraldeído apresentaram os melhores
resultados em termos de conversão após 8 ciclos de reação.
Lipase from Burkholderia cepacia immobilized on superparamagnetic nanoparticles using
adsorption and chemisorption methodologies was efficiently applied as recyclable biocatalyst
in the enzymatic kinetic resolution of (RS)-1-(phenyl)ethanols via transesterification reactions.
(R)-Esters and the remaining (S)-alcohols were obtained with excellent enantiomeric excess
(> 99%), which corresponds to a perfect process of enzymatic kinetic resolution (conversion 50%,
E > 200). The transesterification reactions catalysed with B. cepacia lipase immobilized by the
glutaraldehyde method showed the best results in terms of reusability, preserving the enzyme
activity (conversion 50%, E > 200) for at least 8 successive cycles.
Keywords: superparamagnetic nanoparticles, lipase, transesterification catalysis, enantiomeric
resolution
Introduction
their excellent environmental compatibility, the use of
such superparamagnetic supports represents an effective
green chemistry approach, allowing to extend, through
the successive recovery cycles, the useful lifetime of
the biocatalyst. Among the several synthetic routes to
prepare iron-oxide superparamagnetic nanoparticles,4 the
most employed one for enzyme immobilization is the co-
precipitation methodology which makes use of aqueous
Fe2+/Fe3+ salt solutions. In addition, we can also mention
the use of other magnetic materials5-25 to immobilize
enzymes, such as magnetic metallic nanoparticles,
magnetic microspheres prepared from copolymers with
magnetic particles, hybrid materials (Fe3O4–silica–NiO)
and magnetite-containing mesoporous silica spheres. A
literature survey revealed that several enzymes including
lactase,5 lipase,6 esterase,7 β-galactosidase,8 oxidase,9
dehydrogenase,10 a-chymotripsin,11 chloroperoxidase,12
Penicillin G acylase,13 L-asparaginase,14 tyrosinase,15
horseradish peroxidase,16 chitosanase,17 papain,18 diastase,19
Immobilized lipases have been successfully employed
in hydrolysis1 and trans-esterification catalysis, exhibiting
high enantioselectivity.2 Industrial use of such expensive
biocatalysts suffers yet from a critical point, which
is the lack of efficient enzyme recovery processes.3
This is actually a general problem in catalysis. For
this reason great efforts have been made, pursuing
the best immobilization strategies for the enzymes on
suitable solid supports.3 As a very promising alternative,
superparamagnetic nanoparticles based on magnetite
(g-Fe3O4) can provide outstanding support materials for
the enzymes, exhibiting striking characteristics, such as
large surface area, mobility and high mass transference.
More than this, they can be easily recovered by simple
application of an external magnetic field.4 In addition to
*e-mail: leandroh@iq.usp.br, henetoma@iq.usp.br

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Enzymatic Kinetic Resolution of (RS)-1-(Phenyl)ethanols by Burkholderia cepacia
J. Braz. Chem. Soc.
levansucrase,20 amylase,21 streptokinase,22 dispase,22
dehalogenase,23 laccase24 and epoxide hydrolase25 have been
immobilized onto magnetic particles for different purposes.
In this work, we report on the successful application
of the immobilized form of Burkholderia cepacia lipase
(BCL) on superparamagnetic nanoparticles, exploring
three different immobilization methodologies and their
influence in the enzymatic kinetic resolution of secondary
alcohols.
In this way, theAPTS shell can act as effective binding site
for the lipase protein chains, either via hydrogen bonding
or electrostatic interactions with the amide and aminoacid
residues of the enzyme. Typically, it was employed in the
preparations a protein solution (550 mg L-1; 1 mL) for 20 mg
of APTS-MagNP. After work-out, the amount of protein
immobilized on the superparamagnetic nanoparticles
was determined by Bradford method,31 yielding 0.21 mg
protein/20 mg APTS-MagNP.
The second lipase immobilization method consisted
in the previous modification of the APTS-functionalized
nanoparticles with carboxybenzaldehyde, in order to
make the covalent attachment of the lipase (Scheme 1B).
In the next step, the amino groups from APTS-MagNP
react with the carboxybenzaldehyde, yielding imines,
which can be converted into their secondary amines by
the reduction with NaBH4. Then, the remaining carboxylic
groups from carboxybenzaldehyde moieties can react with
EDC (1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide
hydrochloride), and finally perform the coupling with the
lipase (550 mg L-1). This protocol led to immobilization of
0.23 mg protein on 20 mg Carboxy-APTS-MagNP.
The third immobilization method was based on
glutaraldehyde (Glu) as the coupling agent for making the
covalent attachment of the lipase enzyme to the APTS-
MagNP (Scheme 1C). Glutaraldehyde reacts with the amino
group from APTS-MagNP forming imine bonds, leaving
the terminal aldehyde group for reacting with the amino
residues of the enzyme. In this case, it was possible to bind
0.26 mg protein to 20 mg of Glu-APTS-MagNP.
Results and Discussion
Magnetite (Fe3O4) nanoparticles of about 10 nm
size, were obtained by the co-precipitation method26,27
exhibiting a typical superparamagnetic behavior. In order
to improve their stability and add reactive amino groups
for enzyme immobilization purposes, they were treated
with 3-aminopropyltrimethoxysilane (APTS), generating
APTS-coated nanoparticles, here referred asAPTS-MagNP
(Scheme 1).
The immobilization of B. cepacia lipase on the
superparamagnetic nanoparticles was carried out according
to three different methods28 pursuing the best performance
in the enzymatic kinetic resolution of secondary alcohols.
The first method consisted in the direct interaction
of the enzyme with APTS-MagNP (Scheme 1A). It
should be noticed that at pH 7, lipase from B. cepacia is
negatively charged (isoelectric point = 5.2)29 while the
superparamagnetic nanoparticles exhibit a positive charge
due to the protonation of the aliphatic amines (pKa ca. 9).30
OH
OH
OH
O
O
O
Fe2+
2 Fe3+
Fe3O4
Fe3O4
(i)
(ii)
Si
NH2
APTS-MagNP
(A)
(C)
(B)
(iv,v)
(vii)
(iii)
O
O
O
O
O
O
O
O
O
O
Fe3O4
Fe3O4
Fe3O4
Si
N
Si
OOC
NH3
Si
N
Lipase
H
OH
BCL-APTS-MagNP
Glu-APTS-MagNP
Carboxy-APTS-MagNP
O
(iii)
(vi,iii)
O
O
O
O
O
O
Fe3O4
Fe3O4
Lipase
Si
Si
BCL-Glu-APTS-MagNP
N
N
N
H
N
H
Lipase
O
BCL-Carboxy-APTS-MagNP
Scheme 1. Immobilization of B. cepacia lipase on superparamagnetic nanoparticles (A = Adsorption method; B = Carboxybenzaldehyde method; C
= Glutaraldehyde method); (i) Fe2+/Fe3+ oxides, NaOH (0.5 mol L-1), 0.5 h; (ii) (3-aminopropyl)trimethoxysilane, 12 h; (iii) B. cepacia lipase (550 mg
lipase/mL), 1 h; (iv) 4-carboxybenzaldehyde, 4 h; (v) NaBH4, 1 h; (vi) 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride, 20 min.; (vii)
glutaraldehyde, 2 h.

Vol. 21, No. 8, 2010
Rebelo et al.
1539
O
OH
O
OH
O
Fe3O4
BCL
+
+
O
toluene
R
(R)-2
(S)-1
R
R
(RS)-1
Fe3O4
BCL
R = H (a); MeO (b); Cl (c); Br (d); NO2 (e).
BCL = Burkholderia cepacia lipase
Scheme 2. Enzymatic resolution of (RS)-1-(phenyl)ethanols via enantioselective acetylation catalyzed by B. cepacia lipase.
Enzyme activity: enzymatic kinetic resolution of (RS)-1-
(phenyl)ethanol derivatives
provide a wide range of electron donor and withdrawing
characteristics.
As one can see in Table 1, all reactions yielded the
corresponding (R)-acetates in excellent conversion rates
(50%) with E-value > 200. It is important to mention that
the stereochemistry of the resolved chiral alcohols and
their acetates is in accordance with Kazlauska’s rule.34
Surprisingly, no significant difference between the results
of KR using B. cepacia lipase immobilized by the three
methodologies could be detected.
Inordertoevaluatetherecyclingpotentialofthedifferent
immobilized forms of the B. cepacia lipase on magnetic
nanoparticles [(immobilized by adsorption method (BCL-
APTS-MagNP), by carboxybenzaldehyde method (BCL-
Carboxy-APTS-MagNP) and by glutaraldehyde method
(BCL-Glu-APTS-MagNP)], the KR of (RS)-1a was
carried out in several repetitive reaction cycles (24 h). The
immobilized B. cepacia lipase was collected with a magnet,
and used again in a new experiment (Figure 2).
The lipase activity was determined for the enzymatic
kinetic resolution of (RS)-1-(phenyl)ethanols (1) via
enantioselective acetylation, yielding the corresponding
esters (2) (Scheme 2).
In the control experiments, a sample of B. cepacia
lipase solution, employed in the immobilization studies,
was lyophilized and used in the enzymatic resolution of
(RS)-1-(phenyl)ethanol and (RS)-1-(4-nitro-phenyl)ethanol.
Typical results are shown in Table 1, and can be taken as
reference for comparison purposes with respect to the
catalytic activity of the immobilized enzyme (Figure 1).
As can be seen in Figure 2, the glutaraldehyde
method provided the best lipase reusability, exceeding
8 cycles with the same conversion efficiency (50%) and
enantioselectivity (> 99%), while the other two methods
collapsed after the fifth cycle. These results can be attributed
to the efficient covalent binding of lipase to the magnetic
nanoparticles using glutaraldehyde, which is known as
a good cross-linking agent between enzyme and solid
supports containing amino groups.3 It should be noted that
the long period of each reaction cycle (24 h) employed
in the experiments can be contributing to the decrease of
enzymatic activity. Since the enzymatic kinetic resolution
is carried out in organic solvent, some protein denaturation
can also occur, and consequently, an undesired loss of lipase
activity is observed.3
Figure 1. Comparison of the conversion obtained in the KR of (RS)-1a and
(RS)-1e catalyzed by free (Free BCL) and B. cepacia lipase immobilized
by adsorption (BCL-APTS-MagNP), glutaraldehyde (BCL-Glu-APTS-
MagNP) and carboxybenzaldehyde method (BCL-Carboxy-APTS-
MagNP): (S)-alcohols and (R)-esters (ee > 99%; E > 200).
As one can see in Figure 1, free B. cepacia lipase
afforded the ester 2 with high E-value, but with lower
conversion than in the cases of using immobilized lipase.
This is a remarkable aspect, since besides the possibility
of recycling the biocatalyst, the immobilization process
seems also to improve the enzyme activity, increasing the
conversion rates.
The results from the KR of a racemic mixture
of substituted (RS)-1-(phenyl)ethanols 1a-e using
immobilized B. cepacia lipase are shown in Table 1. The
substituents attached to the aromatic ring were chosen to
Conclusions
Immobilization of lipase from B. cepacia onto
superparamagnetic nanoparticles using different
methodologies afforded a magnetically recoverable, highly
efficient biocatalyst. The lipase activity determined in the
KR of different secondary alcohols afforded excellent

1540
Enzymatic Kinetic Resolution of (RS)-1-(Phenyl)ethanols by Burkholderia cepacia
J. Braz. Chem. Soc.
Table 1. Enzymatic kinetic resolution of (RS)-1-(phenyl)ethanols by B. cepacia lipase immobilized by different methodologiesa
OO
OOHH
OO
OOHH
OO
FFee OO
33 44
BBCCLL
++
++
OO
TToolluueennee
((SS))--11
RR
RR
RR
((RRSS))--11
((RR))--22
BBCCLL ==BBuurrkkhhoollddeerriiaacceeppaacciiaalliippaassee
Entry
Substrate
R
Immobilized form of B. cepacia lipase
ee (%)c
Conversion
Ee
(%)d
(R)-2
(S)-1
1
1a
1b
1c
1d
1e
1a
1b
1c
1d
1e
1a
1b
1c
1d
1e
H
MeO
Cl
BCL-APTS-MagNP
BCL-APTS-MagNP
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
2
3
BCL-APTS-MagNP
4
Br
BCL-APTS-MagNP
5
NO2
H
BCL-APTS-MagNP
6
BCL-Carboxy-APTS-MagNP
BCL-Carboxy-APTS-MagNP
BCL-Carboxy-APTS-MagNP
BCL-Carboxy-APTS-MagNP
BCL-Carboxy-APTS-MagNP
BCL-Glu-APTS-MagNP
BCL-Glu-APTS-MagNP
BCL-Glu-APTS-MagNP
BCL-Glu-APTS-MagNP
BCL-Glu-APTS-MagNP
7
MeO
Cl
8
9
Br
10
11
12
13
14
15
NO2
H
MeO
Cl
Br
NO2
aBCL immobilized by adsorption method (BCL-APTS-MagNP), by carboxybenzaldehyde method (BCL-Carboxy-APTS-MagNP) and by glutaraldehyde
b
method (BCL-Glu-APTS-MagNP); General conditions: Substrates (0.01 mmol), vinyl acetate (30 μL), toluene (1 mL), lipase (adsorption method =
0.21 mg lipase/20 mg APTS-MagNP; carboxybenzaldehyde method = 0.23 mg lipase/20 mg APTS-MagNP; glutaraldehyde method = 0.26 mg lipase/
c
d
20 mg APTS-MagNP), 800 rpm, 24 h, 32 ºC. ee = Enantiomeric excess was determined by chiral GC analysis. Conversion = eeS/(eeS + eeP).
eE = {ln[eeP(1 - eeS)]/(eeP + eeS)}/{ln[eeP(1 + eeS)]/(eeP + eeS)}.32 The absolute configurations of all compounds were determined by comparison of the
sign of the measured specific rotation with those in the literature.33
Figure 2. Reusability of the B. cepacia lipase immobilized by different methodologies (BCL-Carboxy-APTS-MagNP, BCL-Glu-APTS-MagNP and BCL-
APTS-MagNP) in the KR of (RS)-1a to afford the (R)-ester 2a (ee > 99%; E > 200).

Vol. 21, No. 8, 2010
Rebelo et al.
1541
conversion (50%) and enantiomeric ratio (E > 200). The
lipase immobilized by glutaraldehyde method showed the
best results in terms of reusability, preserving the enzyme
activity for at least 8 successive cycles. These results
validate a new green approach to be used in the KR of
secondary alcohols.
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Experimental
General procedure for enzymatic kinetic resolution of
substituted (RS)-1-(phenyl)ethanols (1a-e)
To 2 mL microtube (eppendorff®) containing 20 mg
of magnetic nanoparticles with immobilized lipase by
appropriate methodology (see Table 1), substituted
(RS)-1-(phenyl)ethanols (1a-e) (0.01 mmol) and vinyl
acetate (0.3 mmol) were dispersed in toluene and stirred
with 800 rpm at 32 ºC for 24 h. After the reaction, the
samples were analyzed by GC analysis using a chiral
capillary column.
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Reusability of B. cepacia lipase on the enzymatic kinetic
resolution of (RS)-1-(phenyl)ethanol (1a)
To 2 mL microtube (eppendorff®) containing 20 mg
of magnetic nanoparticles with immobilized B. cepacia
lipase by appropriate methodology (see Figure 2),
(RS)-1-(phenyl)ethanol (1a) (0.01 mmol) and vinyl acetate
(0.3 mmol) were dispersed in toluene and stirred at 32 oC
for 24 h under 800 rpm. After 24 h reaction, the magnetic
nanoparticles were controlled by magnetic and supernatant,
containing the alcohol 1a and ester 2a, was collected for
determination of the enantiomeric excess and conversion.
Then, the immobilized B. cepacia lipase on the magnetic
nanoparticles was washed with toluene (3×1 mL) and then
used for the next cycle.
8. Pan, C.; Hu, B.; Li, W.; Sun,Y.;Ye, H.; Zeng, X.; J. Mol. Catal.
B: Enzym. 2009, 61, 208.
9. Sulek, F.; Drofenik, M.; Habulin, M.; Knez, Z.; J. Magn.
Magn. Mat. 2010, 332, 179; Sohn, O. J.; Kim, C. K.; Rhee, J.
I.; Biotechnol. Bioprocess Eng. 2008, 13, 716; Betancor, L.;
Fuentes, M.; Dellamora-Ortiz, G.; Lopez-Gallego, F.; Hidalgo,
A.; Alonso-Morales, N.; Mateo, C.; Guisan, J. M.; Fernandez-
Lafuente, R.; J. Mol. Catal. B: Enzym. 2005, 32, 97; Kouassi,
G. K.; Irudayaraj, J.; McCarty, G.; J. Nanobiotech. 2005, 3, 1;
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Acknowledgments
The authors acknowledge the support from FAPESP,
CNPq and PETROBRAS.
10. Yusdy; Patel, S. R.; Yapa, M. G. S.; Wang, D. I. C.; Biochem.
Eng. J. 2009, 48, 13; Li, G.Y.; Huang, K. L.; Jiang,Y. R.;Yang,
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Received: December 18, 2009
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