Nanoimmobilization of marine epoxide hydrolase of mugil cephalus for repetitive enantioselective resolution of racemic styrene oxide in aqueous buffer

Article abstract of DOI:10.1166/jnn.2013.7076

We developed two nanoimmobilized biocatalyst systems of thermally unstable Mugil cephalus epoxide hydrolase (McEH) for enantioselective resolution of racemic styrene oxide in aqueous buffer. The recombinant and purified McEH enzyme was immobilized onto ma

Full text of DOI:10.1166/jnn.2013.7076

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All rights reserved
Printed in the United States of America
Journal of
Nanoscience and Nanotechnology
Vol. 13, 2266–2271, 2013
Nanoimmobilization of Marine Epoxide
Hydrolase of Mugil cephalus for Repetitive
Enantioselective Resolution of Racemic
Styrene Oxide in Aqueous Buffer
1
ꢀ†
2ꢀ†
1
1
Young Hyun Kim , Inseon Lee , Sung Hee Choi , Ok Kyung Lee ,
2ꢀ∗
1ꢀ∗
Jungbae Kim , and Eun Yeol Lee
1Department of Chemical Engineering, Kyung Hee University, Gyeonggi-do 446-701, Korea
Department of Chemical and Biological Engineering, Korea University, Seoul 136-701, Korea
2
We developed two nanoimmobilized biocatalyst systems of thermally unstable Mugil cephalus epox-
ide hydrolase (McEH) for enantioselective resolution of racemic styrene oxide in aqueous buffer. The
recombinant and purified McEH enzyme was immobilized onto magnetic nanoparticles (Mag-NPs)
via a two step process of enzyme precipitation and crosslinking. McEH enzyme was also adsorbed,
precipitated, and cross-linked in/on polyaniline nanofibers (PANFs). The residual relative activity of
free McEH, defined as the ratio of residual activity to the initial activity, was 8% after incubation at
ꢀ
3
0 C for 80 h while those of McEH immobilized onto Mag-NPs and in/on PANFs were 15% and 33%
in the same condition, respectively. McEH immobilizations onto Mag-NPs and in/on PANFs could be
Delivered by Publishing Technology to: McMaster University
reused in seven repeti It Pi v :e 1b 8a 2t c. h7 1r e. 1a 0c t9i o. 9n s4 f Oo rn e: n Sa an tt i, o 2s e0 l eF c et i bv e2 h0 y1d 6r o 1l y6s :i 0s 4o :f 4r 4a cemic styrene oxide to
prepare (Sꢀ-styrene oxide Cw oi t hp y9 r8i g% h te: n Aa mn t ieo rmi c ea r ni c Se xc ci ee ns st if (i ce eP) uw bh l ii lse h ree rt sa ining greater than 40–50%
of their initial activity.
Keywords: Magnetic Nanoparticle, Polyaniline Nanofiber, Nanoimmobilization, Epoxide
Hydrolase, Enantioselective Resolution.
1
. INTRODUCTION
advantages such as mild reaction conditions, high selectiv-
ity, low formation of undesired by-products, and catalysis
in aqueous systems. The use of enzymes for the produc-
Drugs with chiral centers are being developed as single
enantiomers. Various synthetic intermediates have been
extensively supplied for the synthesis of chiral pharma-
ceuticals, of which chiral epoxides are one of the most
useful intermediates. Chiral epoxides can be prepared
by chemical methods. Jacobsen’s asymmetric epoxidation
and hydrolytic kinetic resolution technology provide a
commercial process to produce chiral epoxides with 98%
enantiomeric excess (ee) enantiopurity in the presence of
9
1
tion of chiral compounds is one of the most important
research areas in green chemistry. Biocatalytic preparation
of chiral epoxides by epoxide hydrolase (EH) is consid-
2
–5
1
0–12
ered as an emerging alternative to chemical methods.
EH catalyzes the enantioselective hydrolysis of racemic
1
3
epoxides to prepare chiral epoxides.
Commercial implementation of biocatalysis is often
impeded by high costs and instability of enzymes. The
immobilization of enzymes using nanomaterials is pro-
6
ꢀ7
1
mol% catalyst. For these chemical methods, however,
heavy metal-based catalysts and organic solvents were
employed.
1
4–15
posed as a potential solution.
Magnetic nanoparticles
(
Mag-NPs) have been used for various biotechnology
Currently, much effort has focused on applying green
1
6–27
applications,
and the immobilization of enzymes onto
chemistry methods to develop a process that minimizes
the use and generation of hazardous substances.8 Bio-
Mag-NPs in the form of enzyme coatings has been shown
2
8–29
to enhance enzyme stability and reusability.
Glucose
catalysis is a green chemistry approach that offers several
oxidase enzyme was successfully immobilized in and
on polyaniline nanofibers (PANFs) in a three step pro-
cess of enzyme adsorption, precipitation and cross-linking
∗
†
Authors to whom correspondence should be addressed.
These two authors contributed equally to this work.
2
266
J. Nanosci. Nanotechnol. 2013, Vol. 13, No. 3
1533-4880/2013/13/2266/006
doi:10.1166/jnn.2013.7076

Kim et al.
Immobilization of Marine Epoxy Hydrolase for Enantioselective Resolution
(
EAPC), resulting in good enzyme stability as well as high
persulfate, aniline, racemic styrene oxide and (S)-styrene
oxide were purchased from Sigma-Aldrich (St. Louis, MO,
USA). Culture medium components were purchased from
Merck and Difco.
3
0
enzyme loading.
Recently, a novel recombinant EH biocatalyst was
developed by protein engineering of a EH from the
marine fish, Mugil cephalus. A triple-point mutant con-
sisting of F193Y for spatial orientation of the nucle-
ophile, W200L for preventing electron density overlap,
and E378D for good charge relay in the active site
was developed by comparative modeling-inspired site-
3
1
2.2. Cell Culture Conditions and McEH
Recombinant Protein Expression
Recombinant E. coli BL21 (DE3) harboring the recombi-
nant pET-21bꢁ+ꢂ/McEH plasmid containing M. cephalus
3
2
directed mutagenesis. The engineered M. cepahlus EH
McEH), however, is unstable. In the present work, we
(
ꢀ
EH gene was cultured at 37 C and 180 rpm. Expres-
evaluated nano-immobilization methods based on differ-
ent nano-structured supports such as Mag-NPs and PANFs
ꢀ
sion of McEH protein was induced at 15 C by adding
1
mM IPTG to the cell culture and was then maintained
(Fig. 1) and analyzed the stability characteristics of immo-
for 24 h. Cells were harvested at specific time points, and
the specific activity of McEH was analyzed by chiral gas
chromatography (GC).
bilized McEH. The reusability of immobilized McEH was
also investigated for repetitive preparation of enantiopure
ꢁ
Sꢂ-styrene oxide from racemic substrate in an aqueous
buffer.
2.3. Recombinant Protein Purification
2
. MATERIALS AND METHODS
The cells were harvested by centrifugation and resus-
pended in 50 mM sodium phosphate lysis buffer
containing 300 mM NaCl, 10 mM imidazole, 10% (v/v)
glycerol, and 0.5% (v/v) Triton X-100. DNase, lysozyme,
and phenylmethanesulfonyl fluoride were added to final
2
.1. Materials
Sodium phosphate monobasic, sodium phosphate diba-
sic, glutaraldehyde, ammonium sulfate, Tris, ammonium
(
a)
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Copyright: American Scientific Publishers
(
b)
Fig. 1. Schematic representations of nanoimmobilization of M. cephalus epoxide hydrolase (McEH). (a) Enzyme precipitate coating (EPC) onto
magnetic nanoparticles (Mag-NPs) and (b) enzyme adsorption, precipitation and cross-linking (EAPC) in/on polyaniline nanofibers (PANFs).
J. Nanosci. Nanotechnol. 13, 2266–2271, 2013
2267

Immobilization of Marine Epoxy Hydrolase for Enantioselective Resolution
Kim et al.
concentrations of 0.02 mg/mL, 1 mg/mL, and 1 mM,
respectively. The suspension solution was homogenized on
ice with a Vibra Cell VCX 400 ultrasonic processor for
solution (50% w/v). Finally, the enzyme crosslinking was
performed at the GA concentration of 0.5% (v/v) under
tilt-shaking at 4 C for overnight. The capping and wash-
ꢀ
2
2
0 min. The cell debris was centrifuged down (13,000 rpm,
0 min) at 4 C, and the supernatant was filtered using
ing processes was performed in the same way as for the
EPC immobilization onto Mag-NPs. The final sample of
EAPC with McEH in/on PANFs (Fig. 1(b)) was stored at
ꢀ
a 0.2 ꢃm membrane to obtain a clear extract. The result-
ing supernatant containing His-tagged McEH and chaper-
ones was loaded onto a Ni-Sepharose column equilibrated
with 50 mM phosphate buffer (pH 8.0), 0.5 M KCl, and
ꢀ
30
4 C until use.
2.5. Enzyme Activity and Stability Measurements
5
mM imidazole. The column was washed with 30 mL
of the same buffer containing 10 mM imidazole at a flow
rate of 5 mL/min to remove chaperones. Finally, His-
tagged McEH was eluted with the same buffer containing
The measurement of enzyme activity was conducted in
1
mL 100 mM KH PO buffer (pH 7.4) containing the
2 4
enzyme sample. Enantioselective hydrolysis was initiated
by the addition of 10 mM racemic styrene oxide, and the
2
50 mM imidazole. The active fraction was desalted using
ꢀ
™
mixture was shaken at 230 rpm in an incubator at 30 C.
a HiTrap desalting column. The expressed gene prod-
ucts were analyzed by 12% (v/v) SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) as described previously.
The reaction was stopped by the extraction using an equal
volume of cyclohexane.
3
2
The rate of enantioselective hydrolysis and enantiomeric
excess ꢁee = 100 × ꢁꢄSꢅ − ꢄRꢅꢂ/ꢁꢄSꢅ + ꢄRꢅꢂ, where [S]
and [R] represent the concentration of enantiomers, respec-
tively) were analyzed using a GC equipped with a flame
ionization detector (FID) and BETA DEX column (30 m×
2
.4. Preparation of Immobilized McEH
2
.4.1. Enzyme Precipitate Coating (EPC) of McEH on
Magnetic Nanoparticles (Mag-NPs)
0
ꢆ25 mm×0ꢆ25 ꢃm). The temperatures of oven, detector
Iron oxide nanoparticles with average size of 10 nm
were synthesized and further functionalized to graft amino
groups on them as previously reported. Amino group
functionalized Mag-NPs were mixed with enzyme solu-
tion for 2 h. To proceed with the EPC immobilization
technique, the enzyme precipitation was performed in
the ammonium sulfate solution (50% w/v) for 30 min.
Glutaraldehyde treatment (GA) was then performed at a
final GA concentration of 0.1% (v/v) at 4 C under tilt-
ꢀ
and injector were 100, 220 and 220 C, respectively. The
carrier gas was nitrogen in a flow rate of 0.5 mL/min. The
stabilities of free and immobilized McEH were analyzed
by measuring the enzyme activity time-dependently. The
relative activity was calculated from the ratio of residual
3
3
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Copyright: American S ca ice t ni vt ii tf yi c aP tu eb al ci sh h et i rms e point to the initial activity of each
sample. The enzyme samples were incubated at 4, 15, and
ꢀ
3
0 C, respectively.
ꢀ
shaking for overnight. After washing with 10 mM phos-
phate buffer (pH 7.0), unreacted aldehyde groups of GA
were blocked in 100 mM Tris buffer (pH 7.4), and the sam-
ples were excessively washed by using 10 mM phosphate
buffer (pH 7.0). The EPC sample of McEH on Mag-NPs
2
.6. Recycled Uses of Immobilization Enzyme
After each batch of enzyme reaction, the immobilized
McEH onto Mag-NPs or in/on PANFs was separated by
using a magnet or centrifugation at 13000 rpm for 10 min,
respectively. The separated immobilized enzymes were
washed twice with the reaction buffer and then added to a
new reaction solution for repeated batch reactions of enan-
tioselective hydrolysis.
ꢀ
Fig. 1(a)) was stored at 4 C until use.
28ꢀ29
(
2
.4.2. Enzyme Adsorption, Precipitation and
Crosslinking (EAPC) of McEH in and on
Polyaniline Nanofibers (PANFs)
3
. RESULTS AND DISCUSSION
PANFs were prepared by a rapid mixing of monomer and
3
0ꢀ34
initiator as previously reported.
In details, monomer
3.1. Production and Purification of
(aniline) and initiator (ammonium persulfate) were dis-
Recombinant McEH
solved separately in 1 M HCl, and then immediately mixed
and shaken at 200 rpm at room temperature for overnight.
The sample of PANFs was excessively washed with dis-
tilled water, and dried under ambient condition before use.
McEH was immobilized in and on PANFs via a tech-
nique of enzyme adsorption, precipitation, and crosslink-
Recombinant marine EH from M. cephalus (McEH) pos-
sessed high activity and a broad substrate spectrum with
high enantioselectivity for kinetic resolution of racemic
epoxide substrates. However, its stability is low even at
ꢀ
moderate reaction temperatures such as 30 C. In order
3
0
ing as described before. In details, the McEH solution
was mixed with PANFs, and the mixture was shaken at
to enhance the stability of McEH and its reusability in a
repetitive batch mode, we immobilized McEH using nano-
materials such as Mag-NPs and PANFs. In the present
work, the immobilization of McEH onto Mag-NPs or in/on
1
50 rpm for 1 h. After this adsorption step, the enzyme
precipitation was performed in the ammonium sulfate
2
268
J. Nanosci. Nanotechnol. 13, 2266–2271, 2013

Kim et al.
Immobilization of Marine Epoxy Hydrolase for Enantioselective Resolution
3.2. Stability of Immobilized McEH Onto
Mag-NPs and in/on PANFs
McEH was immobilized onto Mag-NPs via an approach of
enzyme precipitate coatings (EPCs), which consists of two
steps: enzyme precipitation and crosslinking (Fig. 1(a)).
On the other hand, McEH was immobilized in/on PANFs
in a three step approach of enzyme adsorption, precipi-
tation and crosslinking (Fig. 1(b)). In order to compare
the stability of free and immobilized McEH, their residual
activity at each time point was measured, and the rela-
tive activity, defined to be the ratio of residual activity to
the initial activity, was used for the stability comparison
of free and immobilized enzymes. First, the stability of
ꢀ
free enzyme was checked at 4, 15, and 30 C. Free McEH
enzyme maintained about 60% of relative activity after
ꢀ
2
20 h incubation at 4 and 15 C. On the other hand, the
Fig. 2. Homology model of McEH protein. The red indicates active site
and green represents the location of lysine residues.
ꢀ
activity of free enzyme rapidly decreased at 30 C, show-
ing a negligible enzyme activity after 120 h of incubation
(
Fig. 4).
PANFs employed glutaraldehyde cross-linking between
amine groups of McEH. The number of lysines, possessing
amine functional groups, in a McEH protein was 32, rep-
resenting the second most abundant amino acid in McEH.
The locations of lysine groups in McEH were identified
by homology-modeling and predicted to be outside the
catalytic active site (Fig. 2). Therefore, we expect that
The stability of McEH immobilized onto Mag-NPs and
ꢀ
in/on PANFs at 4, 15 and 30 C was also investigated.
For McEH immobilized onto Mag-NPs, the relative activ-
ity were 86 and 63% after 220 h of incubation at 4 and
15 C, respectively. For McEH immobilized in/on PANFs,
68 and 67% of the initial activity remained after 220 h
ꢀ
ꢀ
ꢀ
incubation at 4 C and 15 C, respectively. The stability of
both immobilized McEH samples showed reasonably good
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cross-linking between McEH and glutaraldehyde would
not cause a significant loss of catalytic activity.
IP: 182.71.109.94 On: Sat, 20 Feb 2016 16:04:44
ꢀ ꢀ
stability at 4 C and 15 C.
Copyright: American Scientific Publishers
In order to develop McEH biocatalysts immobilized
onto Mag-NPs and in/on PANFs, McEH enzyme was pre-
pared. Recombinant McEH was produced in E. coli in the
presence of chaperones with IPTG induction at exponential
growth phase. The recombinant protein was purified on Ni-
Sepharose columns. The expression level and purification
of McEH protein were analyzed by SDS-PAGE (Fig. 3).
Purified McEH appeared as a single band on SDS-PAGE
with a molecular mass of approximately 45 kDa.
The relative activity of McEH immobilized onto Mag-
NPs decreased to 21 and 5% of the initial activity after
ꢀ
40 and 220 h of incubation at 30 C, respectively (Fig. 5).
For McEH immobilized PANFs, 17% of the initial activity
was retained after 220 h at 30 C. PANF-based immo-
bilization of McEH was more stable than Mag-NP-based
ꢀ
100
8
6
4
2
0
0
0
0
0
0
50
100
150
200
Time (h)
Fig. 3. SDS-PAGE of the purified recombinant McEH protein expressed
in the presence of chaperons (pGro7). Lane 1: whole cells expressing
recombinant McEH proteins, M: molecular marker, lane 2: lysate of the
recombinant cells, lane 3: unbinding protein mixture, lane 4: the purified
recombinant McEH proteins.
Fig. 4. Thermal deactivation of the free McEH enzyme in aqueous
buffer solution (100 mM potassium phosphate, pH 7.5) at each temper-
ꢀ
ature (4, 15 and 30 C). The relative activity was determined from the
ratio of the residual activity at each sampling time to the initial activity.
ꢀ
ꢀ
ꢀ
Symbol: •, 4 C; ꢁ, 15 C; ꢀ, 30 C.
J. Nanosci. Nanotechnol. 13, 2266–2271, 2013
2269

Immobilization of Marine Epoxy Hydrolase for Enantioselective Resolution
Kim et al.
1
00
Table I. Recycling of McEH immobilized onto magnetic nanoparticles
and in/on polyaniline nanofibers. The relative activity was calculated by
the ratio of residual activity to the initial activity after repetitive reactions.
EPC-Mag-NPs and EAPC-PANFs represent magnetic nanoparticle-based
and polyaniline nanofiber-based immobilizations, respectively.
8
6
4
2
0
0
0
0
0
Relative residual activity (%)
Number of enzyme recycling
EPC-Mag-NPs
EAPC-PANFs
1
2
3
4
5
6
7
st
100
91.1
81.1
70.8
46.1
41.9
39.1
100
100
nd
rd
th
th
th
th
74.5
72.8
67.5
58.8
52.8
0
50
100
150
200
Time (h)
of McEH immobilized onto Mag-NPs or in/on PANFs was
above 50% of the initial activity after four and seven rep-
etitions, respectively. This result indicates that nanoimmo-
bilization of McEH onto magnetic nanoparticles or in/on
polyaniline nanofibers enhances both enzyme stability and
reusability in the enantioselective hydrolysis of racemic
styrene oxide in aqueous buffer.
Fig. 5. Prolonged catalytic activity of the nano-immobilized McEH by
EPC onto Mag-NPs and EAPC in/on PANFs. The relative activity was
determined from the ratio of the residual activity at each sampling time to
the initial activity. Symbol: •, free enzyme; ꢁ, EPC-based immobilization
onto Mag-NPs; ꢀ, EAPC-based immobilization in/on PANFs.
immobilization because PANFs provide a porous structure
that would prevent the enzymes from denaturation via con-
finement effect. Although the relative activities of both
types of immobilized McEH was low after 220 h incuba-
4. CONCLUSIONS
ꢀ
tion at 30 C, the stability of immobilized McEH was bet-
Recombinant McEH was immobilized onto Mag-NPs and
in/on PANFs using the EPC and EAPC methods, respec-
ꢀ
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ter than that of free McEH showing a negligible activity in
the same condition. After incubation at 30 C for 80 h, the
residual relative activity of free McEH was 8% while those
of McEH immobilized onto Mag-NPs and in/on PANFs
were 15% and 33% in the same condition, respectively
IP: 182.71.109.94 On: Sat,t 2i v0 e lFy e. bT h2 e0 1r 6e s 1u l6t i: n0 g4 : 4E 4P C and EAPC of McEH showed
Copyright: American Scientific Publishers
enhanced enzyme stability at various temperatures and
under recycled uses due to effective prevention of enzyme
denaturation and leaching. Stabilized McEH in the form
of nanoimmobilization using Mag-NPs and PANFs would
provide a potential use of McEH in the enantioselective
hydrolysis of racemic styrene oxide in aqueous buffer. The
present success of McEH stabilization can be employed
for further improvements of other EH enzymes together
with various enzymes for their successes in applications
where the poor enzyme stability hampers their practical
uses.
(Fig. 5). The immobilized enzymes were more stable than
free enzymes at various temperatures because of effec-
tive enzyme precipitation and crosslinking that inhibit the
enzyme denaturation and leaching. Site-directed mutage-
nesis of McEH using comparative modeling enhanced its
enantioselective hydrolysis activity by 35-fold; thus, only
2
0
a few minutes were required to reach 98% ee. There-
fore, 80 h is sufficient for McEH-catalyzed enantioselec-
tive hydrolysis of racemic styrene oxide in aqueous buffer.
Acknowledgments: This work was supported by the
Marine and Extreme Genome Research Center Program,
Ministry of Land, Transportation and Maritime Affairs.
This work was also supported by the National Research
Foundation of Korea (NRF) grant funded by the Korea
government (MEST) (No. 2012K001385). We thank Drs.
Taeghwan Hyeon and Yong Il Park for the kind gift of
magnetic nanoparticles.
3
.3. Recycling of Immobilized McEH in
Repetitive Batch Mode
Since Mag-NPs have superparamagnetic properties, McEH
immobilized onto Mag-NPs was separated for recycled
uses by using a magnet. McEH immobilized PANFs
could also be separated upon centrifugation. To evalu-
ate the reusability of nanoimmobilized McEHs, the resid-
ual catalytic activity was measured whenever immobilized
McEHs were reused. As the number of reuses increased,
the residual activity gradually decreased (Table I). McEH
immobilized onto Mag-NPs or in/on PANFs was reused
in seven repetitive batch reactions for enantioselective
hydrolysis of racemic styrene oxide for preparing 98% ee
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Received: 18 August 2012. Accepted: 7 November 2012.
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Copyright: American Scientific Publishers
J. Nanosci. Nanotechnol. 13, 2266–2271, 2013
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