Recyclable nanobiocatalyst for enantioselective sulfoxidation: Facile fabrication and high performance of chloroperoxidase-coated magnetic nanoparticles with iron oxide core and polymer shell

Article abstract of DOI:10.1021/ja905477j

(Figure Presented) Magnetic nanoparticles (MNPs) with a core diameter of 30 nm comprising several iron oxide crystals, a poly(glycidyl methacrylate) (PGMA) shell with a thickness of 30 nm, and a surface coated with chloroperoxidase (CPO) were facilely fabricated as a nanobiocatalyst for asymmetric sulfoxidation. The covalently bound CPO did not change the original conformation of the active site and showed the same catalytic activity and enantioselectivity as free CPO for the sulfoxidation of thioanisole to produce (R)-methyl phenyl sulfoxide in >99% ee. The thick PGMA shell significantly increased the stability of the nanobiocatalyst: no loss of the sulfoxidation activity was observed after 11 times of recycling and reuse of the catalyst. Thus, the nanobiocatalyst fabricated here showed the best performance among nanosized biocatalyst particles regarding both the retaining of free enzyme activity and the recycling of catalyst. This is also the first example of a nanobiocatalyst for asymmetric oxidation, and the concept could be generally applicable for fabricating active and recyclable nanobiocatalysts.

Full text of DOI:10.1021/ja905477j

Published on Web 08/24/2009
Recyclable Nanobiocatalyst for Enantioselective Sulfoxidation: Facile
Fabrication and High Performance of Chloroperoxidase-Coated Magnetic
Nanoparticles with Iron Oxide Core and Polymer Shell
Wen Wang,†,‡ Yi Xu,‡ Daniel I. C. Wang,†,§ and Zhi Li*,†,‡
Singapore-MIT Alliance, Department of Chemical and Biomolecular Engineering, National UniVersity of Singapore,
Singapore 117576, and Department of Chemical Engineering, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139
Received July 3, 2009; E-mail: chelz@nus.edu.sg
Magnetic nanoparticles (MNPs) have received increasing atten-
tion in biorelated research due to several distinct properties such
as high surface area to volume ratio, special magnetic behavior,
and high dispersibility in various solvents.1 An important application
for MNPs is biocatalysis which has a profound impact on green
and selective chemical syntheses. Enzyme-coated nanosized mag-
netic particles used as a nanobiocatalyst could be simply recycled
under an external magnetic field. Compared with enzymes im-
mobilized on micrometric supports, a nanobiocatalyst could achieve
a much higher enzyme loading capacity and significantly enhanced
mass transfer efficiency. An attractive approach for fabricating a
nanobiocatalyst is to immobilize the enzyme on iron oxide MNPs
which are cheap and biocompatible. Several enzymes such as
hydrolase,2 glucose oxidase,3 and alcohol dehydrogenase4 have been
thus far immobilized on such MNPs.
Figure 1. Synthetic route of active and stable magnetic nanobiocatalyst.
The major problems in the fabrication of nanobiocatalysts at the
current stage are the unsatisfied catalyst stability during operation
and the recycling process and the dramatically reduced activity in
comparison with free enzyme. Entrapment5 and physical
adsorption2c of enzyme on iron oxide MNPs suffered from the
leakage of enzyme during reaction and recycling (loss of 50%
activity after first cycle2c). Enzymes covalently immobilized on the
surface of iron oxide MNPs showed unsatisfied stability (loss of
55% activity after five runs3), and these particles tended to
aggregate.3,2d The use of silica encapsulated iron oxides MNPs for
covalent enzyme attachment6,2b allowed us to increase the catalyst
stability to some extent (loss of 35% activity after nine runs2b),
but the obtained stability was not sufficient and the activity was
only 22% of its free enzyme activity.2b Furthermore, the silica shell
may contain pores and is unstable under alkaline conditions, the
particle size distribution is difficult to control, and the synthetic
methods are complicated and low-yielding.1b Here we report a new
Figure 2. (a) TEM image of OA-MNPs. (b) TEM image of GMA-MNPs.
(c) FESEM image of EDA-MNPs. (d) FESEM image of CPO-EDA-MNPs.
and simple method for fabricating an active and recyclable
nanobiocatalyst by covalently immobilizing an enzyme on stable
MNPs with a core containing multiple iron oxide MNPs and a thick
polymer shell. Meanwhile, there is an increasing demand to develop
efficient biocatalysts for asymmetric and green oxidations to prepare
enantiopure fine chemicals. In this work, we focus on the first
enantioselective oxidative nanobiocatalyst derived from chlorop-
eroxidase (CPO).
The route for the preparation of the nanobiocatalyst is shown in
Figure 1. Iron oxide particles (OA-MNPs) were prepared in 89%
yield by a coprecipitation method with oleic acid as stabilizer.7 A
TEM image showed a mean size of OA-MNPs of 15 nm in diameter
(Figure 2a), which also indicates that the particles are superpara-
magnetic. Glycidyl methacrylate (GMA) containing an epoxy
functional group was then used as a monomer for in situ polym-
erization in the presence of OA-MNPs, giving 61% of new
nanoparticles (GMA-MNPs) with a core-shell structure and uniform
size distribution (Figure 2b). GMA-MNP has a mean size of ∼90
nm, with a core in a diameter of 30 nm containing several OA-
MNPs and a poly(GMA) shell with a thickness of 30 nm. This
structure could effectively prevent the iron oxide core from leaching
and further oxidation under harsh conditions. For obtaining better
activity of the immobilized enzyme, ethylenediamine was used to
prolong the bridge between the enzyme and MNPs. Reaction of
GMA-MNPs with ethylenediamine gave the corresponding particles
with amino groups (EDA-MNPs) in >99% yield. No dramatic shape
or morphology change was observed during the modification due
† Singapore-MIT Alliance.
‡ National University of Singapore.
§ Massachusetts Institute of Technology.
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10.1021/ja905477j CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
aqueous buffer.11 In contrast, CPO immobilized on other solid
materials resulted in a significant decrease of enzyme activity.9,12
Moreover, the product ee was >99% (R) determined by chiral HPLC
analysis. These results suggest no change in enzyme activity and
enantioselectivity after covalent immobilization. In addition, the
Km of EDA-MNPs-CPO for monochlorodimedon was determined
to be 26.1 µM, similar to the Km of 27.7 µM for free CPO. This
indicated again that there was no significant conformational change
of the enzyme active site after immobilization.
Figure 3. (a) CPO-EDA-MNPs in buffer. (b) Separation of CPO-EDA-
MNPs by magnet after 2 min. (c) VSM of CPO-EDA-MNPs.
Recycling of the nanobiocatalyst was conducted for the sulfoxi-
dation of 5 mM substrate with 5 mM H2O2 for 10 min. After each
cycle, CPO-EDA-MNPs were magnetically separated and added
to the new reaction medium containing substrate and H2O2. The
separation of the particles was easy and high yielding. Figure 4b
showed the concentration of enantiopure (R)-sulfoxide produced
in each cycle. After 12 cycles, the nanobiocatalyst was still fully
active, thus being much better than CPO immobilized on other solid
supporting materials.9,12a
In conclusion, a facile method for preparing MNPs comprising
an iron oxide core, a polymer shell, and an enzyme-coated surface
as a high performance nanobiocatalyst was developed. The co-
valently bound CPO with a long bridge showed the sulfoxidation
activity and enantioselectivity to be the same as those for free CPO.
The thick polymer shell significantly increased the stability of the
nanobiocatalyst, giving no loss of activity after recycling 11 times.
These results are much better than those achieved with CPO on
other solid supports and represent the best performance on activity
retaining as well as catalyst recycling among nanobiocatalysts
known thus far. While it is the first example of a nanobiocatalyst
for asymmetric oxidation, the new concept could be generally
applicable for fabricating active and recyclable nanobiocatalysts.
Figure 4. (a) Sulfoxidation of thioanisole by the nanobiocatalyst and
free CPO. (b) Recycling and reuse of the nanobiocatalyst for the
sulfoxidation.
to the dense polymer coating, and the resulting EDA-MNPs
maintained spherical core-shell structures with a diameter of ∼90
nm (Figure 2c).
An immobilization protocol was demonstrated by using CPO as
the target enzyme. CPO is a versatile peroxidase for chemical
synthesis including asymmetric oxidations, but it requires H2O2 as
the oxidant which often causes significant enzyme deactivation.
CPO has five amino groups from lysine residues, and three of them
are located on the surface opposite to the active site.8 Treatment
of CPO, glutaraldehyde, and EDA-MNPs at pH 4.75 at room
temperature resulted in covalent binding of CPO on the MNPs. A
specific loading of 16.1 mg of CPO/g of MNPs was achieved for
the nanobiocatalyst (CPO-EDA-MNPs) at optimized ratios of those
reactants. This value is five times higher than the reported data
using micrometric support via similar covalent binding.9 From the
FESEM images in Figure 2d, the morphology and size of CPO-
EDA-MNPs did not change significantly after immobilization.
Based on the composition and size of the MNPs, a single EDA-
MNP could be estimated to have a weight of 6.54 × 10-16 g and
a specific surface area of 49 m2 g-1. The Mw of CPO is 50 kDa;
thus ∼126 CPO molecules are bound to one EDA-MNP (see
Supporting Information). This corresponds to 12% occupation of
enzymes on the particle surface area, similar to the value estimated
on the basis of the data for a hydrolase directly immobilized on
iron oxide MNPs.10
The magnetic property of CPO-EDA-MNPs was demonstrated
in Figure 3a and 3b. The nanobiocatalyst was easily and quickly
separated under a magnetic field. Vibrating sample magnetometry
(VSM) in Figure 3c showed that CPO-EDA-MNPs exhibited
superparamagnetic behavior at 298 K with a saturated magnetization
value of 1.74 emu/g of particles.
Asymmetric sulfoxidation of thioanisole to (R)-methyl phenyl
sulfoxide was chosen as the target reaction to investigate the
catalysis and the recycling of CPO-EDA-MNPs. The courses of
the sulfoxidation of 50 mM substrate with 50 mM H2O2 catalyzed
by the nanobiocatalyst and free CPO, respectively, are shown in
Figure 4a. In both cases, the product concentration increased linearly
within 100 min, and no difference in catalytic performance was
observed. The total turnover number of the nanobiocatalyst reached
25 × 103, which is also close to the reported data for free CPO in
Acknowledgment. Financial support from Singapore-MIT Al-
liance through the Flagship Research Projects in Chemical and
Pharmaceutical Engineering Programme is acknowledged.
Supporting Information Available: Procedures for nanobiocatalyst
synthesis, biotransformation, and catalyst recycling; analyses and
characterizations of MNPs; calculations. This material is available free
of charge via the Internet at http://pubs.acs.org.
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