Sputtering deposition of magnetic Ni nanoparticles directly onto an enzyme surface: A novel method to obtain a magnetic biocatalyst

Article abstract of DOI:10.1039/c2cc38737a

A simple one-step method based on the sputtering deposition of Ni nanoparticles (NP) has been developed for the production of magnetic biocatalysts, avoiding the complications and drawbacks of methods based on chemical functionalisation or coating of magn

Full text of DOI:10.1039/c2cc38737a

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Sputtering deposition of magnetic Ni nanoparticles
directly onto an enzyme surface: a novel method to
obtain a magnetic biocatalyst†
Cite this: Chem. Commun., 2013,
4
9, 1273
Received 11th November 2012,
Accepted 23rd December 2012
a
b
b
a
Roberta Bussamara, Dario Eberhardt, Adriano F. Feil, Pedro Migowski,
c
a
d
Heberton Wender, Diogo P. de Moraes, Giovanna Machado,
DOI: 10.1039/c2cc38737a
e
b
a
Ricardo M. Papal e´ o, S e´ rgio R. Teixeira and Jairton Dupont*
www.rsc.org/chemcomm
A simple one-step method based on the sputtering deposition of possibility of achieving very high surface areas, but also because
Ni nanoparticles (NP) has been developed for the production of they provide an easy way to separate enzymes from a reaction
magnetic biocatalysts, avoiding the complications and drawbacks mixture by the application of an external magnetic field. These
1,5–7,10
of methods based on chemical functionalisation or coating of features diminish costs and enhance the product’s purity.
magnetic NP. This new technique provided high levels of recovery, Most of the techniques of enzyme immobilization onto magnetic
reusability and catalytic activity for the lipase–Ni biocatalyst. NP involve modifications of the NP surface in order to chemically
3,6
bind or strongly adsorb the enzyme (Scheme 1aI). Magnetic
Enzymes are versatile biocatalysts that effectively control specific carriers are generally coated with polymeric materials, and these
1
chemical reactions in vivo and in vitro. They present high catalytic over-layers are also modified with various functional groups such as
efficiency and selectivity, operate under mild conditions and hydroxyl, amino or sulfhydryl groups by copolymerization or
2,3
4,7,9
produce fewer by-products than standard chemical catalysts.
Compared with current chemical methodologies, enzymes are
chemical surface modification (Scheme 1aII).
These functional over-layers on magnetic supports can be
environmentally friendlier, more energy-efficient and potentially detrimental to catalytic activity, since multipoint covalent attach-
more cost-effective catalysts due to low energy demand operation ments may promote rigidification of the molecular structure of the
4
5,8
and easier downstream processing. However, biocatalysts immobilized enzyme. Activity loss due to the restriction of con-
composed only of free enzymes are expensive, present poor long- formational changes after immobilization remains a great challenge
term stability under processing conditions and are difficult to to be solved in enzyme engineering, making the development of
2
,3,5
recover and recycle from the reaction mixture.
In order to new immobilization methods one of the main subjects of research
6,7
overcome these deficiencies, various strategies have been employed in this field.
to improve biocatalyst functionality, stability and reusability by
immobilizing enzymes onto carriers through chemical methods.
3
Immobilization of enzymes onto insoluble organic or inorganic
supports maintains the natural catalytic activity and allows bio-
catalyst separation from reaction systems for recycling, to reduce
2,4
operational costs and to mitigate enzyme contamination. A large
number of research reports have shown that immobilized enzymes
1–9
may have better performance than free enzymes in many aspects.
In recent years, the use of magnetic NP for enzyme immobiliza-
tion has received considerable attention not only due to the
a
Instituto de Qu ´ı mica – UFRGS, Av. Bento Gonçalves 9500, 91501-970, Porto
Alegre, RS, Brazil. E-mail: jairton.dupont@ufrgs.br
b
Instituto de F ´ı sica, UFRGS, Porto Alegre, RS, Brazil
c
Laborat o´ rio Nacional de Luz S ´ı ncrotron (LNLS), R. Giuseppe M a´ ximo Scolfaro,
1
0.000, P.O. Box 6192, 13083-970, Campinas, SP, Brazil
Scheme 1 Techniques of enzyme immobilization onto magnetic NP: enzymes
chemically bound or strongly adsorbed on the functionalized surface of the
NP (aI) and enzymes chemically bound on the functionalized surface of the NP coated
with polymeric materials (aII). Decoration of magnetic particles in enzymes by
sputtering (b).
d
e
Centro de Tecnologias Estrat ´e gica do Nordeste, Recife, PE, Brazil
Faculdade de F ´ı sica, PUCRS, Porto Alegre, RS, Brazil
†
Electronic supplementary information (ESI) available: Experimental details. See
DOI: 10.1039/c2cc38737a
This journal is c The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 1273--1275 1273

View Article Online
Communication
ChemComm
Fig. 2 shows a more detailed view of the arrangement of the Ni
NP on the lipase surface. Both TEM (Fig. 2a) and AFM (Fig. 2b)
images indicate the presence of aggregates of Ni particles (black
circular regions in the TEM image and white spots in the AFM
panels) about 50 nm in diameter and a distribution of smaller
particles (B10–20 nm) on top of the amorphous enzyme matrix.
High resolution (HR) TEM of one of these particles (Fig. 2c)
revealed an inter-planar distance of 0.23 nm, corresponding to
the (010) plane of the Ni lattice, which was also confirmed by the
Fourier transform of the HRTEM image. The micrographs in
Fig. 2a and b also demonstrate the uniform dispersion of the Ni
particles, achieved even at the sub-micrometer scale. The quantity
of Ni deposited onto the lipase, determined by flame atomic
absorption spectrometry, was 1.1 mg of nickel for 1 g of the
magnetic biocatalyst. As shown below, this amount was sufficiently
low to allow the substrate access to the enzyme’s active site, but
high enough for the recovery of the enzyme using a magnetic field.
The magnetization curve of the catalyst measured at room
temperature using a superconducting quantum interference device
Fig. 1 Optical images showing the lipase powder before (a) and after (b) sputtering
deposition of Ni. SEM image and EDS of the product after deposition of Ni
(c). Mapping for Ni (d).
In this work, a new approach was developed that enables
1
1–14
enzymes to be decorated with magnetic NP
through a
(SQUID) magnetometer is shown in Fig. 3a. The free lipase showed
simple physical procedure allowing biocatalyst recovery and
reuse in the process. The technique used was the sputtering
a diamagnetic response, as expected. The lipase–Ni composite
magnetization curve showed typical ferromagnetic hysteresis with
a well-defined ‘‘loop’’. Since the lipase mass does not contribute to
magnetization, the Ni content obtained by atomic absorption was
used to estimate the saturation magnetization of the biocatalyst;
1
5
deposition of magnetic particles directly onto the enzyme
surface, without the need for any chemical coating or modifica-
tion of the NP surface. The performance of a nanocomposite
biocatalyst (lipase from Pseudomonas cepacia decorated with Ni
particles) produced by this method was investigated. Excellent
retention of the particles on the enzyme and good levels of
functional activity were obtained, even after several cycles of
use, as discussed below. Details of magnetic biocatalyst
preparation and characterization can be found in detail in ESI.†
Fig. 1a and b show optical images of the lipase powder
before and after sputtering deposition of Ni. As can be seen, the
whitish color of the lipase became dark after the deposition of
Ni. In order to confirm the presence of nickel on the lipase,
scanning electron microscopy (SEM) and energy dispersive
X-ray spectroscopy (EDS) analyses were carried out on the
processed samples (Fig. 1c and d). The Ka characteristic
emission line of Ni at about 7.4 keV could be seen in the
EDS. Ni was uniformly distributed on the lipase surface as
evidenced by the chemical mapping (Fig. 1d).
À1
this was found to be around 4.0 emu g . For bulk Ni at room
À1 16
temperature, the saturation magnetization is 54.4 emu g
. Such
a notable reduction in magnetization compared to the bulk value is
often observed in NP due to the large percentage of surface spins
17
that have a disordered magnetization orientation. In addition, the
À1
data showed a remanence of 1.9 emu g (about 47% of saturation)
and a coercive field of about 58 Oe. Since both values are small but
non-zero, these results imply that the mean size of the NP is above
the critical diameter for superparamagnetism (around 15 nm for
16
Ni), in accordance with the TEM and AFM findings. Note that
sample magnetization saturated with a relatively small external
magnetic field (less than 1000 Oe), meaning that strong magnets
are not required to recycle the biocatalyst, Fig. 3b.
Fig. 3 Magnetization curve of the biocatalyst as a function of the external
magnetic field (a). Magnetic biocatalyst dispersed in aqueous solution being
recycled for reuse by applying a magnetic field (b).
Fig. 2 TEM (a) and AFM (b) images of the lipase after Ni deposition. HRTEM of
one of these Ni NP (c).
1
274 Chem. Commun., 2013, 49, 1273--1275
This journal is c The Royal Society of Chemistry 2013

View Article Online
ChemComm
Communication
Table 1 Hydrolysis activity of the biocatalyst
released into the aqueous medium after hydrolysis was observed
only after the first two recycles (4.1%). The thermal stability of our
catalytic system (see ESI†) was studied and its activity was found to
be maintained up to 70 1C of incubation temperature. Moreover,
we have found that the catalytic system maintains 78% of its
activity after 10 months of storage at 4 1C whereas the free lipase
maintains only 52% of its original activity. Therefore it is probable
that the Ni itself is not the main responsible for the de-activation
processes and the de-activation is related to other phenomena as
suggested for related systems such as inhibition of the active site
a
Batch
Hydrolytic activity Relative
Ni content
activity (%) (mg of Ni)
À1
reactions Biocatalyst (U g of lipase)
—
1
2
3
4
5
6
7
8
9
Free lipase 339
Lipase + Ni 232
Lipase + Ni 230
Lipase + Ni 224
Lipase + Ni 220
Lipase + Ni 203
Lipase + Ni 185
Lipase + Ni 150
Lipase + Ni 139
Lipase + Ni 102
—
100
99.6 Æ 0
—
2.2
oLOQ
96.6 Æ 1.4 oLOQ
94.6 Æ 1.7 oLOQ
87.5 Æ 0.4 oLOQ
79.7 Æ 0.7 oLOQ
64.7 Æ 3.2 oLOQ
60.0 Æ 2.7 oLOQ
44.1 Æ 0.9 oLOQ
27.4 Æ 3.4 oLOQ
5,8
by the reaction media or even enzyme unfolding. Finally it is
important to note that the magnetization properties of the catalytic
system are not affected by its re-use.
1
1
0
1
Lipase + Ni
Ni
63
0
—
—
a
Limit of quantification (LOQ) 0.12 mg of Ni.
We have shown that a simple one-step physical deposition
method can be applied for the production of magnetic bio-
catalysts, avoiding the complications and drawbacks of methods
based on chemical functionalisation or coating of magnetic NP.
This new technique provided high levels of recovery, reusability
and catalytic activity for the lipase–Ni system in both hydrolytic
and synthetic reactions. Such behavior suggests that the sputter
deposition method prevents, to a large extent, structure stiffening
after immobilization, allowing the conformational changes
necessary for the transition from the inactive to the active form
of the enzyme. This new approach can be further improved and
easily expanded to other kinds of magnetic NP and enzymes.
It may provide a simple and inexpensive strategy for the
production of efficient and recyclable magnetic biocatalysts
with potential use on the industrial scale.
Table 2 Transesterification activity of the biocatalyst
a
Batch
Total
Relative
Ni content
reactions Biocatalyst
conversion (%) activity (%) (mg of Ni)
—
1
2
3
4
Free lipase
99
—
100
97.4
87.2
85.9
—
Lipase + Ni 78
Lipase + Ni 76
Lipase + Ni 68
Lipase + Ni 67
oLOQ
oLOQ
oLOQ
oLOQ
—
5
Ni
0
—
a
Limit of quantification (LOQ) 0.12 mg of Ni.
Experiments were performed to investigate the hydrolytic and
transesterification catalytic activity of the pristine and recycled
magnetic biocatalyst (Tables 1 and 2). The magnetic biocatalyst
Thanks are due to CNPq, MCT-INCT, CAPES and FAPERGS.
À1
Notes and references
retained 85% of its hydrolytic activity (232 U g of lipase) and
7
9% of its transesterification activity (78% total conversion)
compared with the free enzyme. For the hydrolytic reaction, over
0% of the residual activity was retained after eight repeated
1 B. Sahoo, S. K. Sahu and P. Pramanik, J. Mol. Catal. B: Enzym., 2011,
6
9, 95–102.
2
X. Jin, J. F. Li, P. Y. Huang, X. Y. Dong, L. L. Guo, L. Yang, Y. C. Cao,
F. Wei, Y. D. Zhao and H. Chen, J. Magn. Magn. Mater., 2010, 322,
2031–2037.
6
batch reactions (Table 1). Concerning the transesterification
reaction, over 85% of the activity was retained after five repeated
batch reactions (Table 2). Moreover, only in the first run a small
amount of Ni (2.19 mg) was released into the aqueous medium
after hydrolysis and in the organic phase after transesterification
with the reuse of the magnetic biocatalyst (Tables 1 and 2).
Our results were also compared to data from the literature in
which enzymes were chemically immobilized onto magnetic
particles. Lipase from Candida rugosa immobilized onto
Fe O magnetic NP supported in ionic liquids via adsorption
3
4
5
6
Y. Yong, Y. X. Bai, Y. F. Li, L. Lin, Y. J. Cui and C. G. Xia, J. Magn.
Magn. Mater., 2008, 320, 2350–2355.
M. M. Zheng, L. Dong, Y. Lu, P. M. Guo, Q. C. Deng, W. L. Li,
Y. Q. Feng and F. H. Huang, J. Mol. Catal. B: Enzym., 2012, 74, 16–23.
C. L. Pan, B. Hu, W. Li, Y. Sun, H. Ye and X. X. Zeng, J. Mol. Catal. B:
Enzym., 2009, 61, 208–215.
Y. Y. Jiang, C. Guo, H. S. Xia, I. Mahmood, C. Z. Liu and H. Z. Liu,
J. Mol. Catal. B: Enzym., 2009, 58, 103–109.
7
8
B. Shu, G. Zheng, L. Wei and S. Yan, Food Chem., 2006, 96, 1–7.
S. Mohapatra, N. Panda and P. Pramanik, Mater. Sci. Eng., C, 2009,
29, 2254–2260.
9 S. Y. Shaw, Y. J. Chen, J. J. Ou and L. Ho, Enzyme Microb. Technol.,
2006, 39, 1089–1095.
3
4
6
presented 64% immobilization efficiency.
Functionalized Fe O magnetic NP were used to immobilize
10 Y. Pan, X. W. Du, F. Zhao and B. Xu, Chem. Soc. Rev., 2012, 41,
3
4
2912–2942.
3
lipase by electrostatic adsorption and covalent binding, retaining 11 T. P. N. Ngo, W. Zhang, W. Wang and Z. Li, Chem. Commun., 2012,
8, 4585–4587.
2 M. Lin, D. Lu, J. Zhu, C. Yang, Y. Zhang and Z. Liu, Chem. Commun.,
012, 48, 3315–3317.
4
70% of its hydrolytic activity. These results suggest that covalent
1
bond formation via epoxy groups and amino groups reduces the
2
conformational flexibility. A 37% decrease in immobilized enzyme 13 S. Y. Lee, S. Lee, I. H. Kho, J. H. Lee, J. H. Kim and J. H. Chang,
Chem. Commun., 2011, 47, 9989–9991.
4 K. S. Lee, M. H. Woo, H. S. Kim, E. Y. Lee and I. S. Lee, Chem.
Commun., 2009, 3780–3782.
3 4
activity was also observed for an esterase immobilized on Fe O
magnetic NP via a glutaraldehyde coupling reaction. Covalent
1
9
attachment promotes rigidification of the molecular structure of 15 F. Endres and S. Z. El Abedin, Chem. Commun., 2002, 892–893.
5,8
16 Y. W. Du, M. X. Xu, W. Jian, Y. B. Shi, H. X. Lu and R. H. Xue, J. Appl.
Phys., 1991, 70, 5903–5905.
the immobilized enzyme. In our work, the magnetic biocatalyst
retained 85% of its hydrolytic activity and the remaining activity
was about 80% after five reuses. The amount of total protein
1
7 R. H. Kodama, A. E. Berkowitz, E. J. McNiff and S. Foner, Phys. Rev.
Lett., 1996, 77, 394–397.
This journal is c The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 1273--1275 1275