Immobilization of burkholderia cepacia on pristine or functionalized multi?walled carbon nanotubes and application on enzymatic resolution of (RS)?1?phenylethanol

Article abstract of DOI:10.21577/0103-5053.20180063

The immobilization of Burkholderia cepacia lipase (BCL) on pristine or functionalized multi-walled carbon nanotubes (MWCNTs) was studied in the resolution of (RS)-1-phenylethanol. For the functionalization, three treatments were used, these being in H

Full text of DOI:10.21577/0103-5053.20180063

http://dx.doi.org/10.21577/0103-5053.20180063
J. Braz. Chem. Soc., Vol. 29, No. 9, 1876-1884, 2018
Printed in Brazil - ©2018 Sociedade Brasileira de Química
Article
Immobilization of Burkholderia cepacia on Pristine or Functionalized
Multi‑Walled Carbon Nanotubes and Application on Enzymatic Resolution
of (RS)‑1‑Phenylethanol
a
a
a
a
Michele R. G. Dias, Alysson de Pauloveloso, Lilian F. M. do Amaral, Rhaísa T. Betim,
b
,a
Maria G. Nascimento and Cristiane Pilissão*
a
Departamento de Química e Biologia, Universidade Tecnológica Federal do Paraná,
1280-340 Curitiba-PR, Brazil
8
b
Departamento de Química, Universidade Federal de Santa Catarina,
8040-900 Florianópolis-SC, Brazil
8
The immobilization of Burkholderia cepacia lipase (BCL) on pristine or functionalized multi-
walled carbon nanotubes (MWCNTs) was studied in the resolution of (RS)-1-phenylethanol. For
the functionalization, three treatments were used, these being in H SO /HNO (acid, MWCNTs-A),
2
4
3
KOH (basic, MWCNTs-B) or in H O (oxidizing agent, MWCNTs-O). It was found that the
2
2
pristine or functionalized BCL/MWCNTs resulted in a 3-fold improvement in the conversion and
in a 5-fold improvement in ee (enantiomeric excess of the substrate), when compared with free
s
BCL under the same reaction conditions. This pristine or functionalized BCL/MWCNTs system
could be reused for up to 8 times without significant decrease in the catalytic efficiency. Under the
optimum conditions (pH of immobilization 6.0, 45 ºC and 25 mg/5 mg pristine or functionalized
BCL/MWCNTs), the best results were obtained using BCL immobilized on MWCNTs-A. The
simple process of physical adsorption of BCL onto MWCNTs-A has improved the catalytic
efficiency when compared with free BCL and an increase in the stability was confirmed by
thermogravimetric analysis (TGA).
Keywords: multi-walled carbon nanotubes, functionalization, Burkholderia cepacia lipase,
immobilization, enzymatic resolution
Introduction
Carbon nanotubes (CNTs), amongst various types of
nanomaterials, can have their properties altered by further
functionalization on their surface. This functionalization
can be either on their walls, tips, or by encapsulating them
and it may bring substantially different properties compared
with the non-functionalized nanotubes. Their chemically
modified structures can be used to facilitate the interaction
of nanotubes with organic molecules, such as enzymes.
Several studies were conducted to change multi-walled
Enzyme immobilization can improve the performance
of an enzyme, increasing its stability and resistance to a
wide range of pH and temperature. Therefore, it preserves
the enzyme activity through several catalytic cycles.
However, the process to select an adequate support is
intricate and depends on the type of enzyme, reaction
media, reaction conditions, and safety policy of the field
1
-5
2
of application. Moreover, in order to make this process
economically feasible, it is important to take into account
some factors such as the type of support and method of
immobilization, since they influence activity and reuse of
carbon nanotubes (MWCNTs). MWCNTs can be oxidized
using several oxidative agents such as acids (nitric and
sulfuric acid, HNO /H SO ), hydrogen peroxide (H O )
3
2
4
2
2
1
2
and base (sodium hydroxide, NaOH). The oxidation
of MWCNTs occurs particularly at defective sites such
as ring defects, edges, dangling bonds and kink sites. In
the oxidation process, several functional groups such as
carboxyl (–COOH) and hydroxyl (–OH) are formed on the
surface of nanotubes. Some oxidants preferably form acidic
groups (–COOH) while others preferably form “basic”
groups (–OH) as a result of their different properties.
4
-9
the biocatalyst.
Recently, nanomaterials such as carbon nanotubes,
nanoparticles, and nanofibers have been increasingly
explored as support for enzyme immobilization due to
2,4,5,10,11
their exclusive properties and various applications.
*
e-mail: pilissao@utfpr.edu.br

Vol. 29, No. 9, 2018
Dias et al.
1877
These functional groups can be designed according to the
application.
Thus, in this work, the effect of MWCNTs
functionalization and their application as support for the
immobilization of Burkholderia cepacia lipase (BCL) were
studied in the resolution of (RS)-1-phenylethanol (1) with
vinyl acetate. Several experimental conditions such as pH of
immobilization, the temperature of reaction, mass support/
lipase ratio and reaction time were evaluated. Reuse of
immobilized BCL was also analyzed (Scheme 1).
1
3-15
MWCNTs have quickly risen as a support for enzyme
immobilization because of their high surface area,
minimum diffusion limitations, maximum amount of
enzyme loading, high mechanical stability, mobility and
16,17
high mass transference.
Immobilization of enzyme on MWCNTs can be
achieved by covalent and non-covalent approaches; the
latter is preferred since it is carried out without chemical
additives, preserving the native conformation of the
Experimental
2
,10,18,19
enzyme.
surface of MWCNTs can occur by different mechanisms:
i) adsorption by van der Waals interactions and formation
Non-covalent binding of the enzyme on the
Chemicals
(
Multi-walled carbon nanotubes purchased from Sigma-
Aldrich (purity ≥ 98%, 10 ± 0.1 nm × 4.5 ± 0.5 nm ×
3-6 µm (o.d. × i.d. × length)) were used without any further
treatment (pristine MWCNTs). Sodium borohydride, boric
acid, acetic anhydride (97%) and vinyl acetate (99%) were
purchased from Vetec. Lipase from Burkholderia cepacia
of π–π stacks between aromatic residues of enzymes and the
MWCNTs surface; (ii) hydrophobic interactions between
hydrophobic side chains of amino acids and the MWCNTs
surface; (iii) amphiphilic binding generating a surfactant-
like interaction; (iv) electrostatic interactions in which both
the isoelectric point of the enzyme and the point of zero
charge of MWCNTs play a role; and (v) hydrogen bonding
between amine terminal groups of enzymes and oxygen-
-1
(BCL, 30000 U g ), previously known as Pseudomonas
cepacia, was donated by Amano Pharmaceutical Co.
Other reagents used for the enzymatic resolution were of
analytical grade and were obtained commercially.
19-24
containing groups in oxidized MWCNTs.
Lipases are the class of enzymes that shows the highest
degree of hydrophobicity with 28-30% of hydrophobic
amino acid residues. Furthermore, those hydrophobic
residues are usually close to the active site which make them
prone to activation after being adsorbed on hydrophobic
Synthesis and characterization of standards
(RS)-1-Phenylethanol was obtained by the reduction
of acetophenone using sodium borohydride and boric acid
according to the methodology described by Cho et al.
2,16
26
supports like MWCNTs.
Because of their ability to catalyze esterification and
transesterification reactions in organic solvents, lipases
(S)-1-Phenylethanol was obtained from acetophenone
using Daucus carota roots, according to the methodology
described by Omori et al. (RS)-1-Phenylethyl acetate
was synthesized using the following methodology: acetic
anhydride (25 mmol), (RS)-1-phenylethanol (5 mmol)
2
7
(
Enzyme Commission (EC) No. 3.1.1.3) have been
showing several applications in food and pharmaceutical
2
5
industries.
Scheme 1. Kinetic resolution of (RS)-1-phenylethanol using BCL immobilized on pristine or functionalized MWCNTs (BCL/MWCNTs).

1
878
Immobilization of Burkholderia cepacia on Pristine or Functionalized Multi-Walled Carbon Nanotubes
J. Braz. Chem. Soc.
and sulfuric acid (1%) were dissolved in 30 mL of
dichloromethane. The solution was refluxed for 6 h. The
reaction mixture was cooled to room temperature and
washed with 5% sodium bicarbonate solution (40 mL),
and then with water (40 mL). The organic layer was dried
using sodium sulfate. The compounds obtained here were
used as standard compounds for the gas chromatography
analysis.
Immobilization of BCL on pristine and functionalized
MWCNTs by physical adsorption
Pristine MWCNTs and functionalized MWCNTs-F,
MWCNTs-A, MWCNTs-B and MWCNTs-O (5 mg)
were suspended in 5 mL potassium phosphate buffer
-
1
(50 mmol L ) for pH 6.0-8.0 or potassium phosphate
-
1
buffer (135 mmol L ) for pH 5.0 and 5.5, containing
BCL (5-50 mg). The mixture was incubated at 25 ºC
for 24 h with constant stirring at 150 rpm. Then, the
suspension was centrifuged for 10 min (3000 rpm) to
remove the supernatant. The derived immobilized lipase
(pristine or functionalized BCL/MWCNTs) was dried
overnight at 30 ºC and ground into a powder, which was
used in the kinetic resolution of 1 with vinyl acetate. The
immobilization of BCL was further confirmed by FTIR
spectroscopy and thermogravimetric analysis (TGA). The
experimental data were obtained in triplicate.
The esters were also characterized with aVarian-640-IR
Fourier transform infrared (FTIR) spectrometer using
attenuated total reflectance (ATR) for liquid samples
-1
1
(
2
4000-650 cm ). The H NMR spectra were recorded at
00 MHz with a Bruker DPX 200 spectrometer using
CDCl as the solvent and tetramethylsilane (TMS) as the
3
internal standard at Central Analítica, Departamento de
Química, Universidade Federal do Paraná (UFPR, Curitiba-
PR, Brazil). The pH of immobilization and pH used in the
functionalized MWCNTs (MWCNTs-F) were measured
using a Marte MB10 pHmeter.
Characterization of pristine and functionalized MWCNTs
Functionalization and purification of MWCNTs
Pristine MWCNTs, functionalized MWCNTs and
BCL/MWCNTs were characterized in a ratio of 1:1000
mass of sample/KBr by FTIR. The spectra were obtained
using a Bomem spectrometer (model Varian-640-IR)
For the functionalization of the pristine MWCNTs,
three treatments were used: acid, basic and oxidizing agent.
-
1
Acid treatment
in transmission mode between 4000 and 400 cm at a
-1
Pristine MWCNTs (20 mg) were mixed with 10 mL
resolution of 4 cm .
-
1
nitric acid (3.0 mol L ) and 10 mL sulfuric acid
The TGA analyses were performed on SDT Q600
instrument (TA Instruments) from 50 to 900 ºC in a
-1
(
3.0 mol L ), and refluxed for 6 h. Then, the MWCNTs
3
-1
functionalized by acid treatment (MWCNTs-A) were
separated from the solution by centrifugation (3000 rpm)
and washed with a solution of water/ethanol (1:1 v/v) until
pH 7.0. The sample was dried overnight at 50 ºC.
nitrogen atmosphere (flow rate: 20 cm min ; heating rate:
-1
10 ºC min ) using samples less than 10 mg.
Determination of protein loading
Basic treatment
The protein loading was determined by means of the
Bradford method in which the free BCL powder contains
1 wt.% protein.
Pristine MWCNTs (100 mg) were mixed with 100 mL
ethanol and 5 g potassium hydroxide, and refluxed for
28
8
h. Subsequently, the MWCNTs functionalized by basic
The amount of lipase bound to pristine MWCNTs or
functionalized MWCNTs-F was determined indirectly
from the difference between the amount of lipase
(concentration of protein in the free BCL) introduced
into the reaction mixture and the amount of lipase in the
filtrate (final protein concentration) after immobilization
measured by the Bradford method, using bovine serum
albumin (BSA) as the standard. All experiments were
performed in triplicate.
treatment (MWCNTs-B) were separated from the solution
by centrifugation (3000 rpm) and washed with a solution
of water/ethanol (1:1 v/v) until pH 7.0. The sample was
dried overnight at 50 ºC.
2
8
Oxidizing treatment
Pristine MWCNTs (20 mg) were mixed with 80 mL
3
0% hydrogen peroxide solution and refluxed for 2 h.
Then, the MWCNTs functionalized by oxidizing treatment
MWCNTs-O) were separated from the solution by
The immobilization yield was determined following
3
(
equation 1:
centrifugation (3000 rpm) and washed with a solution of
water/ethanol (1:1 v/v) until pH 7.0. The sample was dried
overnight at 50 ºC.
(
1)

Vol. 29, No. 9, 2018
Dias et al.
1879
A yield of immobilization of 98% was achieved after
4 h of immobilization at room temperature.
The immobilized BCL on pristine MWCNTs/BCL or
functionalized MWCNTs-F/BCL powder was used in the
resolution of 1 with vinyl acetate to evaluate the catalytic
efficiency of this system in this reaction.
Effect of lipase loading
2
The influence of the amount of free BCL or BCL
immobilized on pristine MWCNTs was evaluated in the
resolution of (RS)-1 with vinyl acetate using n-hexane at
35 ºC for 24 h, and pH of immobilization 7.0 (potassium
phosphate buffer). The amount of pristine BCL/MWCNTs
used was in the range of 5-50 mg in 5 mg of pristine
MWCNTs and 25 mg of free BCL. The results of
General procedure for lipase-catalyzed resolution of
(RS)-1-phenylethanol
conversion degrees and ee are presented in Figure 1.
s
The reactions were carried out in 15 mL pure n-hexane
containing 1 mmol racemic (RS)-1-phenylethanol, 1 mmol
vinyl acetate and 5-50 mg free or immobilized BCL.
The reactions were performed in a 125 mL stoppered
flask at 25-65 ºC in a Dubnoff water bath at 150 rpm
for 3-24 h. Aliquots were withdrawn at specified time
intervals from the reaction mixture, and then analyzed
by chiral chromatography. The enantiopreference
of the formed product was compared to the chiral
(
S)-alcohol standard by gas chromatography. The
conversion and the enantiomeric excesses of the formed
products were determined with a gas chromatograph
(
(
GC-14B, Shimadzu) equipped with a chiral column
RT-BetaDEX-sm, 30 m × 0.32 mm × 0.25 µm, Agilent).
Figure 1. Influence of free BCL or BCL immobilized on pristine
MWCNTs on the enzymatic resolution of (RS)-1-phenylethanol with
vinyl acetate: () conversion and () ee . Reaction conditions: (RS)-1
H was used as the carrier gas with a pressure of 120 kPa
s
2
(
1 mmol), vinyl acetate (1 mmol), n-hexane (15 mL), 24 h, 35 ºC, pH of
-1
and flow rate of 1.40 mL min . The temperature of the
injector and detector was 230 ºC. The column temperature
was an isotherm of 100 ºC for 30 min. The retention times
observed for S-(–) and R-(+)-1-phenylethanol were 19.4 and
immobilization 7.0 (potassium phosphate buffer, 50 mmol), 25 mg of free
BCL or 5-50 mg BCL/5 mg pristine MWCNTs.
As it can be observed, the conversion degrees increased
from 4 to 28% as the amount of BCL increased until
2
0.9 min, respectively, and for the corresponding S-(–) and
R-(+)-acetyl esters were 17.6 and 19.2 min, respectively.
25 mg. The ee values were in the range of 4-38% and
s
The enantiomeric ratio (E) was calculated from the
ee > 99% (data not shown), resulting in E > 200, and
p
enantiomeric excess of the product (ee ), enantiomeric
the enantiopreference was for the (R)-1, thus forming the
R-ester. When the reaction was carried out using 50 mg of
p
excess of the substrate (ee ), and the conversion degree
s
29
(
c) according to the method described by Chen et al. An
BCL, no improvement in the conversion degree or ee was
s
experiment was performed with pristine MWCNTs in the
absence of BCL, and no product was detected.
observed. Therefore, 25 mg of BCL were selected to be
used in the following studies.
Results and Discussion
Effect of pH on BCL immobilization on pristine MWCNTs
Enzymes are highly affected by pH, both during
3
1
Effect of lipase loading, pH and temperature
immobilization and along biocatalysis processes.
Changing the pH will alter the surface charge of both the
enzyme and the MWCNTs, hence affecting the electrostatic
interactions between enzyme and support.
It is well known that the immobilization conditions
have significant effects on the immobilization efficiency.
3
0
1,32
Conditions such as lipase loading-support, pH of
immobilization and temperature were firstly evaluated in
the transesterification reaction of (RS)-1-phenylethanol
with vinyl acetate using the system BCL/MWCNTs.
When the optimal condition was defined, the effect of
functionalized MWCNTs on the resolution was also
studied.
Thus, the effect of pH on the immobilization of BCL
on pristine MWCNTs was evaluated in the resolution of
the (RS)-1 in the range of 5.0 to 8.0. The data of conversion
degrees ee and ee are given in Figure 2.
As can be observed in Figure 2, using BCL immobilized
on pristine MWCNTs, the best results were achieved at
s
p
pH 6.0, where the conversion degree was of 48%, ee 91%
s

1
880
Immobilization of Burkholderia cepacia on Pristine or Functionalized Multi-Walled Carbon Nanotubes
J. Braz. Chem. Soc.
2
4
The same behavior was observed by Pereira et al.,
using Candida rugosa lipase immobilized on chitosan in
the synthesis of n-butyl butyrate. The pH decreased from
7
.0 in the free form to 6.0, after immobilization. Fadiloglu
34
and Soylemez also observed a reduction in the optimal
pH from 7.0 to 6.5 after C. rugosa lipase immobilization on
celite. From these results, pH 6.0 was selected to be used
to evaluate the other parameters, such as the influence of
temperature and the functionalization of MWCNTs.
Effect of temperature
Figure 2. Effect of pH on the immobilization of BCL and its influence
on the enzymatic resolution of (RS)-1-phenylethanol with vinyl acetate
mediated by pristine BCL/MWCNTs. () conversion and () ees.
Reaction conditions: (RS)-1 (1 mmol), vinyl acetate (1 mmol), pristine
BCL/MWCNTs (25 mg/5 mg), n-hexane (15 mL), 35 ºC, 24 h.
Another key factor that may affect the rate of a reaction
catalyzed by an enzyme is the temperature. The temperature
influences the activity, selectivity and stability of the
35
biocatalyst besides the reaction equilibrium.
Thus, to evaluate this parameter in the resolution of
the (RS)-1 with vinyl acetate catalyzed by the system
BCL/MWCNTs, the temperature was changed from 35
to 65 ºC. The results showed no significant changes in the
conversion degrees, being 50% in this temperature range.
and ee > 99%, resulting in E > 200. Using pH of 6.5
and 6.8, a small decrease in the conversion degrees and
p
ee values was observed, of 42 and 41, and 72 and 69%,
s
respectively. Using pH in the range of 7.0-8.0, a large
decrease in both conversion degrees and ee values was
The selectivity showed small changes, being ee > 99%
s
s
observed, of 12-27 and 13-37%, respectively. However,
and ee from 86 to > 99%, resulting in E-values > 200.
p
no change was observed in the ee values (> 99%). When
These data are interesting and show the high stability of the
immobilized BCL on pristine MWCNTs. Based on these
data, a temperature of 45 ºC was considered as appropriate
to evaluate the influence of MWCNTs functionalization.
p
pH 5.0 and 5.5 were used, the conversion degrees were
low, of 1 and 10%, ee of 0.5 and 24%, but with ee > 99%,
s
p
resulting in E-value > 200.
The results may be related to the interactions of
the support and lipase surface, and in this case, can be
considered satisfactory for an enzymatic resolution. It
is usually described in the literature that the pH of the
lipase in free form may be different than immobilized
MWCNTs functionalization effect on BCL immobilization in
the resolution of (RS)-1-phenylethanol
The effect of MWCNTs surface chemistry on the
efficiency of the immobilization of Burkholderia cepacia
lipase by simple non-covalent adsorption was explored in
the resolution of (RS)-1-phenylethanol with vinyl acetate.
1
,22,24,32
33
form.
As previously reported by Lou et al.,
the optimum pH of the free BCL is 7.0. However, it
is interesting to note that in this study, a change in the
optimum pH value from 7.0 to 6.0 was observed after
immobilization on MWCNTs.
The results of conversion degrees and ee are presented in
s
Table 1.
Table 1. Effect of functionalization of MWCNTs in the immobilization of BCL and application in the resolution of (RS)-1-phenylethanol
Reaction time / h
entry
Support/BCL
3
6
12
24
c / %
8 ± 1
ee / %
c / %
12 ± 2
36 ± 3
45 ± 2
38 ± 2
40 ± 3
ee / %
c / %
17 ± 2
42 ± 2
46 ± 1
42 ± 3
43 ± 2
ee / %
c / %
24 ± 3
45 ± 4
47 ± 2
43 ± 2
44 ± 1
ee / %
s
s
s
s
1
2
3
4
5
free BCL
pristine MWCNTs
MWCNTs-A
MWCNTs-B
9 ± 1
50 ± 2
72 ± 2
57 ± 5
46 ± 2
13 ± 2
69 ± 5
87 ± 2
59 ± 1
62 ± 2
20 ± 3
79 ± 1
88 ± 2
66 ± 1
70 ± 2
31 ± 5
82 ± 13
87 ± 5
73 ± 3
76 ± 2
33 ± 3
41 ± 3
35 ± 3
32 ± 1
MWCNTs-O
BCL: Burkholderia cepacia lipase; c: conversion degree; ee : enantiomeric excess of the substrate; MWCNTs: multi-walled carbon nanotubes; MWCNTs-A,
s
MWCNTs-B and MWCNTs-O: MWCNTs functionalized by acid, basic and oxidizing treatment, respectively. Reaction conditions: (RS)-1 (1 mmol),
vinyl acetate (1 mmol), free BCL (25 mg), MWCNTs (25 mg BCL and 5 mg of pristine and functionalized MWCNTs (MWCNTs-A, MWCNTs-B, and
MWCNTs-O)), n-hexane (15 mL), 45 ºC, immobilization pH 6.0.

Vol. 29, No. 9, 2018
Dias et al.
1881
Four types of MWCNTs (pristine MWCNTs,
MWCNTs-A, MWCNTs-B and MWCNTs-O) were used to
immobilize BCL. When pristine or functionalized MWCNTs
were used, the results were better than those obtained using
free BCL. With their use as catalysts, the conversion degrees
were 32-47%, ee > 99%, ee of 46-88%, resulting in E > 200
of the back-bone and side-chain of polar amino acids present
on the outer surface of the BCL protein.
17
The difference in conversion degrees and selectivity
values using the pristine or functionalized MWCNTs may
be related to the interaction between both phases of the
lipase and support. These interactions depend on the surface
and properties of the support, and these properties may
be of primary importance in enzyme catalyzed reactions,
since they are capable of affecting the conformation of the
p
s
in 3-24 h of reaction. Using free BCL, the conversion degrees
were 8-24%, ee > 99% and ee of 9-31% in the same time.
p
s
These values represent an increase of more than 3-fold in
2,16
the conversion and 5-fold in the ee . These results showed
enzymes and, consequently, their reactivity.
s
high activity catalytic and selectivity of the BCL when used
in immobilized form in the resolution of (RS)-1.
The hydrophobic surface of pristine MWCNTs
provides a suitable microenvironment for lipase, where
the interaction among BCL and MWCNTs can be through
π–π stacks interactions, as well as by hydrophobic
The best results were obtained using BCL immobilized
on MWCNTs-A, forming the R-ester in conversion degrees
of 41-47% in 3-24 h of reaction, ee 72-88% and ee > 99%,
19-21
interactions. The possibility of surface functionalization
s
p
resulting in E > 200. After 6 h of reaction, no significant
increase was observed in both conversion degrees and
of pristine MWCNTs can modify their properties, and thus
improve the interaction between enzyme and support.
16,17,36
ee values. Similar results were obtained when BCL was
immobilized on pristine MWCNTs in 24 h of reaction. The
main product was the (R)-3.
The results presented in this work showed this
improvement in catalytic activity and stability when the
carbon nanotube was functionalized in acid medium,
hence, showing a better interaction between BCL and
MWCNTs-A.
s
The chemical functionalization of nanotubes has been
intensely analyzed to attach to the surface of the tube
active chemical groups that can anchor through covalent or
non-covalent bonds other groups or molecules. Among the
various groups used for functionalization, the carboxylic
group stands out. In this work the best results were obtained
when BCL was immobilized on MWCNTs-A. In the acid
treatment (H SO /HNO ) the polar groups (COO–) are
Reuse of BCL/MWCNTs system
One of the advantages of the immobilization is the
improvement in stability and activity of the enzymes.
Likewise, the reuse of the system is another advantage of the
immobilization. In this study, the BCL/MWCNTs systems
were used in the resolution of (RS)-1-phenylethanol with
vinyl acetate in 6 h. After each reaction, the immobilized
lipase was separated, washed with n-hexane, desiccated in
vacuum and then reused for 8 successive cycles. The results
are presented in Table 2.
2
4
3
introduced into the non-polar surface and at the ends of the
support MWCNTs. Thus, the surface of the MWCNTs is
fixed to other polar portions (NH , OH) present in the BCL
2
protein. Therefore, the role of the MWCNTs is to anchor the
BCL protein to the MWCNTs through the oppositely charged
carboxyl moiety (electron rich) and hydrogen (electron poor)
Table 2. Reuse of free or immobilized BCL on pristine or functionalized MWCNTs using different methodologies in the resolution of (RS)-1-phenylethanol
Free BCL
MWCNTs
MWCNTs-A
MWCNTs-B
MWCNTs-O
Cycle
c / %
12 ± 2
6 ± 1
6 ± 2
2 ± 2
2 ± 2
0 ± 0
0 ± 0
0 ± 0
ee / %
c / %
36 ± 3
45 ± 3
35 ± 2
36 ± 2
41 ± 1
40 ± 4
36 ± 3
33 ± 5
ee / %
c / %
ee / %
c / %
ee / %
c / %
ee / %
s
s
s
s
s
1
2
3
4
5
6
7
8
13 ± 2
69 ± 5
45 ± 2
50 ± 2
41 ± 3
46 ± 2
50 ± 2
49 ± 1
48 ± 1
46 ± 1
87 ± 2
38 ± 2
44 ± 2
33 ± 3
37 ± 4
42 ± 2
37 ± 0
37 ± 0
36 ± 1
59 ± 1
40 ± 3
48 ± 3
37 ± 3
38 ± 3
42 ± 4
40 ± 2
34 ± 3
29 ± 1
62 ± 2
6 ± 1
7 ± 3
2 ± 2
2 ± 2
0 ± 0
0 ± 0
0 ± 0
76 ± 1
54 ± 4
56 ± 4
70 ± 1
75 ± 4
56 ± 5
51 ± 7
99 ± 2
68 ± 4
85 ± 5
98 ± 3
94 ± 3
92 ± 2
83 ± 5
78 ± 6
50 ± 4
64 ± 2
75 ± 7
58 ± 1
59 ± 1
56 ± 5
82 ± 5
59 ± 4
61 ± 5
73 ± 4
69 ± 5
52 ± 6
42 ± 1
BCL: Burkholderia cepacia lipase; MWCNTs: multi-walled carbon nanotubes; MWCNTs-A, MWCNTs-B and MWCNTs-O: MWCNTs functionalized
by acid, basic and oxidizing treatment, respectively; c: conversion degree; ee : enantiomeric excess of the substrate. Reaction conditions: (RS)-1 (1 mmol),
s
vinyl acetate (1 mmol), free BCL (25 mg), MWCNTs (25 mg BCL and 5 mg of pristine and functionalized MWCNTs (MWCNTs-A, MWCNTs-B, and
MWCNTs-O)), n-hexane (15 mL), 45 ºC, 6 h, immobilization pH 6.0.

1
882
Immobilization of Burkholderia cepacia on Pristine or Functionalized Multi-Walled Carbon Nanotubes
J. Braz. Chem. Soc.
As can be observed in Table 2, using free BCL, the
The MWCNTs-A spectrum (Figure 3a) showed some
-1
conversion degrees to (R)-3 were 2-12%, ee of 2-13%
characteristic bands. The band at 3435 cm corresponds
to –OH stretching vibration of the surface groups, and
s
and ee > 99% (data not shown). These results showed a
p
-1
decrease in the catalytic activity and selectivity after the
first reaction cycle.
another at 1631 cm originating from conjugated –C=C–
-
1
bonds. The band at 1719 cm is due to the carbonyl
stretch of the carboxylic group, where the conjugation
of C=O with C=C results in a lower vibration frequency
When the pristine MWCNTs or functionalized
MWCNTs-F(MWCNTs-A, MWCNT-B, andMWCNTs-O)
systems were used, better results were achieved. No
significant change in the conversion degrees, ee and ee ,
1
7
-1
of carbonyl group. The band at 1103 cm is assigned to
C–O stretching vibrations, confirming the oxidation of
s
p
2
3 10,17
values was observed after 8 reaction cycles. The conversion
degrees were 29-50%, ee of 42-99% and ee > 99% (data
sp hybridized carbon in pristine MWCNTs to sp .
The
-
1
absorption bands in the region of 2850-2970 cm refer
to the asymmetric stretching of aliphatic C–H bonds. It
is clearly observed that all characteristic bands of the
protein are present in the spectrum of the immobilized
enzyme, which undoubtedly confirmed that this process
was successful.
s
p
not shown in Table 2). The results herein obtained indicated
that BCL did not show any deactivation during the reaction
or washing procedure. Therefore, these results can be
attributed to the immobilization of BCL on pristine or
functionalized MWCNTs, which maintained the catalytic
activity and stability during the reuse. These results yield,
as expected, another advantage of the process: its simplicity
and scalability to industrial proportions can be explored
economically.
The free lipase spectrum (Figure 3b) is typical for
-
1
a protein, with the most prominent band at 1643 cm
from –C=O stretching and –NH bending vibrations and
-
1
a band at 1021 cm , which was attributed to the C–N
bond, thus confirming the presence of the amide. The
-
1
Characterization of functionalized MWCNTs and immobilized
BCL/MWCNTs
band at 3394 cm was due to amide stretching (N–H),
which confirms the presence of this group. Differences
in the spectra of free BCL and BCL immobilized on
MWCNTs-A (Figure 3c) are significant in the 1386-1457
BCL immobilized on functionalized MWCNTs-A was
confirmed and characterized by FTIR and TGA and their
corresponding details will be described in the following
sub-sections.
-1
and 900-1150 cm regions.Also, after immobilization, the
band due to the vibration of –OH group shifted from 3435
-1
to 3415 cm , indicating that the hydroxyl groups formed
2
hydrogen bonds with the lipase.
FTIR analysis
The successful immobilization of lipase onto
MWCNTs-A was confirmed by FTIR spectroscopy.
FTIR spectra of MWCNTs-A, free BCL and immobilized
BCL/MWCNTs-A are presented in Figure 3.
Thermogravimetric analysis
The thermogravimetric method is often used to monitor
any reaction that involves oxidation or dehydration. By
measuring the decomposition or weight loss of the sample,
TGA reveals the change in thermal stability of the support
used in the immobilization process, indicating if it has
37
been modified. It is well known that different structural
forms of carbon can exhibit different oxidation behaviors
depending each time on the available reactive sites. Herein,
the thermal decomposition of functionalized MWCNTs-A,
as well as free and immobilized BCL on MWCNTs-A, was
investigated. This analysis was performed in the range of
5
0-900 ºC, and the data is presented in Figure 4.
In this study, TGA was used as a tool to judge the
thermal stability of immobilization of BCL on MWCNT-A.
It was observed that free BCL was totally decomposed
at 50-480 ºC, in which 15 wt.% was below 70 ºC, due to
evaporation of the water present in the lipase. When the
temperature increased until 150 ºC, the sample showed a
large degradation (45 wt.%), which may be associated with
elimination of water molecules present in close vicinity
Figure 3. FTIR spectra of (a) raw MWCNTs-A, (b) free BCL and
(
c) immobilized BCL/MWCNTs-A (KBr = 0.1%).

Vol. 29, No. 9, 2018
Dias et al.
1883
efficiency and enantioselectivity of the immobilized lipase
was observed, under the optimum conditions (pH 6.0 of
immobilization, 45 ºC and a BCL/MWCNTs molar ratio
of 25 mg/5 mg). The best results were obtained using BCL
immobilized on MWCNTs-A, forming the R-ester (3) in
conversion degrees of 45-47%, ee of 82-87% and ee > 99%
s
p
(E > 200), in 6-24 h of reaction. After 6 h, no significant
increase in the conversion degrees was observed. The
pristine or functionalized lipase/MWCNTs systems could
be reused for 8 cycles without significant decrease in the
catalytic efficiency. In summary, the MWCNTs present
great advantages as supports for BCL immobilization and
offer potential advantages for the applications in (RS)-1
resolution.
…
Figure 4. Termogravimetric analysis for () MWCNTs-A, ( ) free
BCL and (----) BCL/MWCNTs-A. Experimental conditions: nitrogen
3
-1
-1
atmosphere, flow rate: 20 cm min , heating rate: 10 ºC min .
Supplementary Information
of the enzyme. It had totally decomposed at temperatures
between 150-480 ºC. Similar results were observed by
Turner and Vulfson.
Supplementary data are available free of charge at
http://jbcs.sbq.org.br as PDF file.
38
The MWCNTs-A started to decompose around 50 ºC
with a weight loss of approximately 10 wt.%. This mass
loss can be related to the evaporation of adsorbed water. The
second stage of decomposition, from 50-550 ºC, is attributed
to the decarboxylation of the carboxylic groups and by the
elimination of hydroxyls present on the MWCNTs wall and
are totally burned when the temperature reaches 667 ºC.
A different behavior was observed for the BCL/MWCNTs
system, in which three stages of decomposition were
observed. The first was between 50-200 ºC, the second at
Acknowledgments
This work was supported by Universidade Tecnológica
Federal do Paraná (UTFPR), Pós-Graduação em Química
(PPGQ)-Universidade Federal de Santa Catarina (UFSC),
Conselho Nacional de Desenvolvimento Científico
e Tecnológico (CNPq, process No. 476159/2013-0),
Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES), and Laboratório Multiusuários
de Análise Químicas (LAMAQ-UTFPR). We also
thank Amano Pharmaceutical Co. (Japan) for the
donation of BCL, Prof Hugo A. Gallardo (UFSC) for
the termogravimetric analysis, technician Rubia Bottini
(UTFPR), and Departamento de Química da Universidade
Federal do Paraná for the NMR analysis.
200-250 ºC and the third in the range of 250-580 ºC. In this
temperature range, the mass loss was 50 wt.%. Moreover, it
can be clearly seen that thermal stability of the immobilized
lipase is considerably higher as compared to the free form.
As described, free BCL presented a mass loss of 45 wt.%
up to 150 ºC, and after immobilization (BCL/MWCNTs-A)
the mass loss was 7% up to 200 ºC and 43 wt.% up to
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reused in 8 consecutive cycles, maintaining high catalytic
activity (see data in Table 2).
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Published online: March 23, 2018
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