Reversible clustering of magnetic nanobiocatalysts for high-performance biocatalysis and easy catalyst recycling

Article abstract of DOI:10.1039/c2cc30953j

Reversible clusters of nanobiocatalysts are developed via non-covalent interaction among enzyme-bound iron oxide magnetic nanoparticles. Dissociation of the clusters by shaking during biotransformation enables high catalytic performance, and re-clustering

Full text of DOI:10.1039/c2cc30953j

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2012, 48, 4585–4587
www.rsc.org/chemcomm
COMMUNICATION
Reversible clustering of magnetic nanobiocatalysts for high-performance
biocatalysis and easy catalyst recyclingw
Thao P. N. Ngo, Wei Zhang, Wen Wang and Zhi Li*
Received 9th February 2012, Accepted 7th March 2012
DOI: 10.1039/c2cc30953j
Reversible clusters of nanobiocatalysts are developed via non-covalent
interaction among enzyme-bound iron oxide magnetic nanoparticles.
Dissociation of the clusters by shaking during biotransformation
enables high catalytic performance, and re-clustering by stopping
shaking after reaction allows for easy magnetic separation. The novel
concept is demonstrated with alcohol dehydrogenase RDR for
the enantioselective reduction of 7-methoxy-2-tetralone.
giving the final catalyst with too large size and no advantages
of nanobiocatalysts. It is a dilemma in the development of
nanobiocatalysts: while high enzyme loading and high catalytic
performance require small size of catalyst, easy and complete
separation needs big size of catalyst.
Herein, we report a novel concept and practical method to
solve this challenging problem via reversible clustering of
magnetic nanobiocatalysts. In this concept, nanobiocatalysts
with high enzyme loading are designed to form reversible
clusters of micro-size via non-covalent interaction among
enzyme-bound MNPs (Fig. 1); the clusters are easily dissociated
into individual nanobiocatalysts in reaction medium by gentle
shaking to catalyze the desired transformation with high
performance; and the nanobiocatalysts quickly form clusters
again after reaction by stopping shaking, allowing for easy,
fast, and complete separation of the catalysts.
Immobilization of enzymes on magnetic nanoparticles (MNPs)
as nanobiocatalyst has received increasing attention for in vitro
biotransformation, since nanobiocatalysts could achieve higher
enzyme loading due to the higher surface area to volume ratio as
well as higher catalytic performance due to higher mass transfer
efficiency in reaction medium. Many enzymes were immobilized
1–4
on the cheap and biocompatible iron oxide MNPs, and some
of them achieved good activity and stability. However, despite
the special magnetic behaviour of MNPs, facile recycling of
nanobiocatalysts remains a significant challenge in practical
application.
The route for preparing the reversibly clustered magnetic
nanobiocatalysts is outlined in Fig. 1. GMA-MNPs with a
diameter of 62 nm (TEM) containing multiple iron oxide
MNPs (7 nm, TEM) as core, poly(glycidyl methacrylate)
Iron oxide MNPs behave as superparamagnets only when
their diameter is below 15 nm, and it is impossible to effectively
capture a single MNP of this size even by high gradient magnetic
5
separation (HGMS). One approach to solve this problem is to
coat multiple sub-15 nm iron oxide MNPs with a polymer to
4,6
form a bigger MNP with stable core–shell structure. Attaching
enzymes on such MNPs with a diameter of 100–200 nm was
4
reported to give good catalytic performance, and the recycling
of the nanobiocatalysts was also demonstrated. However, long
separation time and strong magnetic field are required for
nanobiocatalysts of these sizes, and it is difficult to achieve
complete separation. On the other hand, clustering of sub-15 nm
7
iron oxide MNPs by other methods as nanoclusters is known,
and a diameter of >500 nm was thought to be suitable for
8
separation under low to moderate magnetic field strength.
Fabrication of biocatalysts with such nanoclusters requires further
coating, functionalization, and immobilization with enzymes,
Department of Chemical and Biomolecular Engineering,
National University of Singapore, 4 Engineering Drive 4, Singapore,
117576, Singapore. E-mail: chelz@nus.edu.sg; Fax: +65 67791936;
Tel: +65 65168416
w Electronic supplementary information (ESI) available: Experimental
details on the synthesis, characterization, stability, biocatalysis, and
recycling of the reversibly clustered nanobiocatalysts. See DOI: 10.1039/
c2cc30953j
Fig. 1 Synthesis and biocatalysis of reversibly clustered magnetic
nanobiocatalysts.
This journal is c The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 4585–4587 4585

View Article Online
routinely used in practical biotransformation was enough to
dissociate the clusters. After stopping shaking, the nanoparticles
clustered again within 3 min to give a mean size of 4.8 mm.
Immobilization of His-tagged RDR with CHO-MNPs at a
pH of 5.5 in KP buffer (7 mM) did not give RC RDR-MNPs,
but formed fine nanobiocatalysts (RDR-MNPs) with a diameter
of 65 nm (FESEM). Adjusting the pH to 8 resulted in the fast
clustering of RDR-MNPs within 5 min. This clearly demonstrated
that a pH of 8 is essential for the clustering. At this pH,
CHO-MNPs have zero charge, which gives insufficient electrostatic
repulsion to counteract the attraction and aggregation caused by
van der Waals forces. The presence of enzymes on MNPs is
also necessary for the clustering: shaking of CHO-MNPs in
KP buffer (0 to 90 mM) at a pH of 8 in the absence of enzymes
did not result in any clusters. The enzymes could contribute to
the clustering by the attractive forces between enzymes
through permanent and induced dipoles. Moreover, salts help
the clustering by decreasing the hydration layer around the
enzymes and increasing hydrophobic interactions between
enzymes. At a pH of 8.0, immobilization of His-tagged RDR
with CHO-MNPs in KP buffer at a KP concentration lower
than 2 mM did not give RC RDR-MNPs. However, increase
of KP concentration to 7 mM resulted in fast formation of the
clusters of nanobiocatalysts.
Fig. 2 (a) TEM of CHO-MNPs. (b) Optical microscopy of RC
RDR-MNPs. (c) FESEM of RC RDR-MNPs. (d) Optical microscopy
of RC RDR-MNPs after shaking at 300 rpm and 30 1C for 2 min.
(
PGMA) as shell, and epoxy group as surface function were
4
prepared as the starting material. 4,7,10-Trioxa-1,13-tridecane-
diamine was reacted with GMA-MNPs to give NH -MNPs
2
containing amine groups on the surface, followed by the
reaction with glutaraldehyde to afford CHO-MNPs containing
aldehyde surface groups. CHO-MNPs were found to have a
diameter of 63 nm by TEM (Fig. 2a) and a hydrodynamic size
of 157 nm by DLS (pH 7.0). Importantly, CHO-MNPs have
zero charge at a pH of 8 (Fig. S5, ESIw), providing the
possibility of forming clusters after enzyme immobilization.
The size of CHO-MNPs at this pH is 255 nm (DLS), small
enough to achieve high enzyme loading and high mass transfer
efficiency. The long bridge between the aldehyde group and the
surface of CHO-MNPs could contribute to retaining the confor-
mation and activity of the free enzyme. Alcohol dehydrogenase
The easy separation of RC RDR-MNPs under a magnet is
demonstrated in Fig. 3a and b: complete separation was
achieved within only 4 s. In comparison, the separation of fine
RDR-MNPs under a magnet was not complete even after 20 min
(Fig. 3c and d), as evidenced by the brown yellow colour of the
solution that contains some nanoparticles. RC RDR-MNPs
are superparamagnetic with a saturated magnetization of
11 emu per g particles, similar to the fine RDR-MNPs, shown
in the vibrating sample magnetometry (VSM) of Fig. 3e and f.
Accordingly, the spins on neighboring particles inside the
clusters were not close enough to influence the magnetization
9
RDR (Molecular weight of 28 KD) from Devosia riboflavina was
chosen as a model enzyme for demonstrating the concept, since it
is a useful enzyme for the enantioselective reduction of ketones
to prepare enantiopure alcohols. To immobilize the enzyme,
1
1
per mass of particles.
The catalytic performance of RC RDR-MNPs was examined
with enantioselective reduction of 7-methoxy 2-tetralone to
produce (R)-7-methoxy-2-tetralol (Fig. 4a), a useful and valuable
1
0
His-tagged RDR was shaken with CHO-MNPs in potassium
phosphate (KP) buffer (7 mM) at a pH of 8 and 4 1C for
1
2
4
h, which resulted in reversibly clustered RDR-MNPs
pharmaceutical intermediate. As the enzymatic reduction
is dependent on the expensive cofactor NADH, isopropanol
was used as ‘‘the couple substrate’’ to regenerate the cofactor.
(
RC RDR-MNPs). The overall synthesis of the nanobiocatalyst
clusters is simple and highly reproducible, with 89% yield from
GMA-MNPs, 76% enzyme loading efficiency, and a specific
enzyme loading of 53 mg RDR per g particles. No enzyme was
leached during washing or sonication, suggesting that the
enzyme was covalently bound to the MNPs.
RC RDR-MNPs have a mean size of 8.5 mm (Fig. 2b) and
contain individual nanobiocatalysts with a diameter of 68 nm in
the clusters (Fig. 2c). Sonication of RC RDR-MNPs in KP buffer
for 15 min gave monodispersed individual nanobiocatalysts
with a diameter of 70 nm (Fig. S9, ESIw), suggesting that the
individual nanobiocatalysts in the clusters were not cross-
linked. Dissociation of the clusters was also easily achieved
by shaking the suspension of RC RDR-MNPs in KP buffer at
3
00 rpm and 30 1C for 2 min. As shown in the microscopy
image (Fig. 2d), all big particles disappeared and only several
particles with a size of 200–700 nm were left (particles with a
size less than 200 nm are invisible in the microscopy image due
to the detection limit). Accordingly, a gentle shaking force
Fig. 3 (a and b) Magnetic separation of RC RDR-MNPs: (a) t = 0,
(b) t = 4 s. (c and d) Magnetic separation of fine RDR-MNPs: (c) t = 0,
(d) t = 20 min. (e) VSM of RC RDR-MNPs. (f) VSM of fine RDR-MNPs.
4
586 Chem. Commun., 2012, 48, 4585–4587
This journal is c The Royal Society of Chemistry 2012

View Article Online
under the magnetic field. RC TLL-MNPs were also successfully
recycled 10 times while retaining 92% activity (see ESIw).
A novel concept of reversible clustering of magnetic nano-
biocatalysts for high-performance biocatalysis and easy catalyst
recycling is successfully developed. Nanobiocatalysts form reversible
clusters via interactions between enzymes immobilized on neutrally
charged iron oxide MNPs. The clusters are easily dissociated
into individual nanobiocatalysts by gentle shaking for efficient
biotransformation, and the nanobiocatalysts re-cluster quickly by
stopping shaking for easy, fast, and complete separation under a
magnet. The concept is proven by using an alcohol dehydrogenase
(
RDR). The nanobiocatalyst clusters RC-RDR-MNPs are
prepared in high yields with high enzyme loading and demonstrate
00% activity and the same enantioselectivity of the free enzyme in
bioreduction. They are effectively recycled 14 times to produce
R)-7-methoxy-2-tetralol in >99% ee and 125 mM while retaining
0% of the original productivity. The concept has been proven
1
Fig. 4 (a) Scheme of enantioselective bioreduction of 7-methoxy-2-
tetralone with cofactor recycling. (b) Time course of the biotransformation
at a pH of 8 with RC RDR-MNPs and free RDR, respectively.
(
8
(
c) Recycling of RC RDR-MNPs in the enantioselective bioreduction.
general with T. lanuginosus lipase (TLL) and could open an avenue
for developing practical in vitro biotransformations for different
types of enzymatic reactions for green chemical syntheses.
Financial support from Ministry of Education of Singapore
through an AcRF Tier 1 Grant (Project No.: R-279-000-239-112)
is greatly acknowledged.
RC RDR-MNPs or free His-tagged RDR was used at 0.1 mg
protein per mL for the reduction of 10.5 mM 7-methoxy-2-
tetralone, with the addition of 0.0012 mM NADH and 48 mM
isopropanol, in Tris buffer (6 mM, pH 8) at 300 rpm and 30 1C
for 60 min. As shown in Fig. 4b, RC RDR-MNPs retained
1
00% activity of the free enzyme, which is better than any
3
Notes and references
reported nanobiocatalysts coated with a dehydrogenase.
After 60 min, 7-methoxy-2-tetralol was produced in 97% yield
1
A. Dyal, K. Loos, M. Noto, S. Chang, C. Spagnoli, K. Shafi,
A. Ulman, M. Cowman and R. Gross, J. Am. Chem. Soc., 2003,
(
10.2 mM) with RC RDR-MNPs as the biocatalyst. The
1
25, 1684; J. Lee, Y. Lee, J. Youn, H. Na, T. Yu, H. Kim, S. Lee,
enantioselectivity of RC RDR-MNPs was also the same as
that of the free enzyme, giving the product in >99% ee (R).
Furthermore, NADH was efficiently regenerated for 8500 times,
which is practical for the synthesis of the chiral pharmaceutical
intermediate. These results demonstrated the high performance
of the nanobiocatalyst clusters in biotransformation. They also
indicated no significant change in the active site of the enzyme
before and after immobilization and clustering.
Y. Koo, J. Kwak and H. Park, Small, 2008, 4, 143.
L. Rossi, A. Quach and Z. Rosenzweig, Anal. Bioanal. Chem.,
2004, 380, 606.
K. Goldberg, A. Krueger, T. Meinhardt, W. Kroutil, B. Mautner
and A. Liese, Tetrahedron: Asymmetry, 2008, 19, 1171; M. H. Liao
and D. H. Chen, Biotechnol. Lett., 2001, 23, 1723; M. Liao and
D. Chen, J. Mol. Catal. B: Enzym., 2002, 18, 81.
2
3
4 W. Wang, Y. Xu, D. I. C. Wang and Z. Li, J. Am. Chem. Soc.,
009, 131, 12892; W. Wang, D. I. C. Wang and Z. Li, Chem.
Commun., 2011, 47, 8115.
S. Shylesh, V. Schunemann and W. Thiel, Angew. Chem., Int. Ed.,
2010, 49, 3428; A. H. Lu, E. Salabas and F. Schuth, Angew. Chem.,
Int. Ed., 2007, 46, 1222.
L. P. Ramırez and K. Landfester, Macromol. Chem. Phys., 2003,
04, 22; J. Kim, J. E. Lee, J. Lee, J. H. Yu, B. C. Kim, K. An,
2
Recycling of RC RDR-MNPs was conducted in the same
biotransformation. After 20 min reaction, RC RDR-MNPs
were quickly separated under a magnet, washed, and then reused
for a new cycle of reaction. As shown in Fig. 4c, the catalyst
retained 80% of its original activity after recycling 14 times. This
result is much better than those from any reported dehydrogenases
5
6
¨
¨
´
2
Y. Hwang, C.-H. Shin, J.-G. Park, J. Kim and T. J. Hyeon, J. Am.
Chem. Soc., 2005, 128, 688; H. Xu, L. Cui, N. Tong and H. Gu,
J. Am. Chem. Soc., 2006, 128, 15582; S. Sacanna and A. P. Philipse,
Adv. Mater., 2007, 19, 3824.
R. Sondjaja, T. Alan Hatton and M. K. C. Tam, J. Magn. Magn.
Mater., 2009, 321, 2393; A. Ditsch, P. E. Laibinis, D. I. C. Wang and
T. A. Hatton, Langmuir, 2005, 21, 6006; J.-F. Berret, N. Schonbeck,
F. Gazeau, D. El Kharrat, O. Sandre, A. Vacher and M. Airiau,
J. Am. Chem. Soc., 2006, 128, 1755; J. K. Stolarczyk, S. Ghosh and
D. F. Brougham, Angew. Chem., Int. Ed., 2009, 48, 175; J. Jin,
T. Iyoda, C. Cao, Y. Song, L. Jiang, T. J. Li and D. B. Zhu, Angew.
Chem., Int. Ed., 2001, 40, 2135.
1
3
immobilized on solid supports. In total, 125 mM of (R)-7-
methoxy-2-tetralol was produced in >99% ee and 80% yield,
by recycling NADH 6000–7700 times in each cycle.
7
RC RDR-MNPs showed much higher tolerance against lower
pH than the free enzyme and retained nearly the same productivity
even at pH 5.5 (Fig. S15a, ESIw). They were also much more
thermostable than the free enzyme. After 12 h pre-incubation at
7
0 1C, the free enzyme dropped by 95% in productivity, while
RC RDR-MNPs lost only 6% of their original productivity
Fig. S15b, ESIw). These improved stabilities are probably due
8
Yusdy, S. R. Patel, M. G. S. Yap and D. I. C. Wang, Biochem. Eng. J.,
2
009, 48, 13; M. Franzreb, M. Siemann-Herzberg, T. J. Hobley and
O. R. T. Thomas, Appl. Microbiol. Biotechnol., 2006, 70, 505.
9 N. Kizaki, T. Nishiyama and Y. Yasohara, US patent 20070178565,
007.
(
to the covalent attachment of enzymes on the nanocarriers.
The generality of the concept was demonstrated by using
Thermomyces lanuginosus lipase (TLL). The reversible cluster
RC TLL-MNPs were fabricated in 89% yield, with 42 mg enzyme
per g particles and a mean size of 27 mm. They were easily
dissociated by shaking, showing 93% activity of the free enzyme for
hydrolysing p-nitrophenyl butyrate. Clusters were quickly formed
after stopping shaking and completely separated within 4 s
2
1
1
0 W. L. Tang, Z. Li and H. Zhao, Chem. Commun., 2010, 46, 5461.
1 D. R. Ingram, C. Kotsmar, K. Y. Yoon, S. Shao, C. Huh, S. L. Bryant,
T. E. Milner and K. P. Johnston, J. Colloid Interface Sci., 2010, 351, 225.
12 M. Mogi, K. Fuji and M. Node, Tetrahedron: Asymmetry, 2004,
5, 3715.
3 Z. D. Zhou, G. D. Li and Y. J. Li, Int. J. Biol. Macromol., 2010,
7, 21; G. Y. Li, K. L. Huang, Y. R. Jiang, D. L. Yang and
P. Ding, Int. J. Biol. Macromol., 2008, 42, 405.
1
1
4
This journal is c The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 4585–4587 4587