Carboligation reactions mediated by benzoylformate decarboxylase immobilized on a magnetic solid support

Article abstract of DOI:10.1002/chir.22181

In this study, magnetic nanoparticles (Fe₃O₄, magnetite) with immobilized metal affinity ligands (MSS) were prepared and characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FTIR), and vibrating-sample magnetometer (VSM) methods for purification and immobilization of the histidine-tagged recombinant benzoylformate decarboxylase (BFD). The MSS support was shown to be eligible for selective binding of HIS-tagged BFD by SDS-page analysis. Loading capacity of the MSS support was determined as 43.6 ± 1.1 mg/g. The regeneration ability for protein binding was also studied. An immobilized BFD was tested to catalyze benzoin condensation and representative cross acyloin reaction. Conversion and enantiomeric excess values were comparable with that of free enzyme catalyzed reactions. Chirality 25:415-421, 2013.

Full text of DOI:10.1002/chir.22181

CHIRALITY (2013)
Carboligation Reactions Mediated by Benzoylformate Decarboxylase
Immobilized on a Magnetic Solid Support
1
1
BILSEN TURAL, SERVET TURAL, AND AYHAN S. DEMIR2
*
1Department of Chemistry, Dicle University, Diyarbakir, Turkey
2Department of Chemistry, Middle East Technical University, Ankara, Turkey
ABSTRACT
In this study, magnetic nanoparticles (Fe3O4, magnetite) with immobilized metal
affinity ligands (MSS) were prepared and characterized by X-ray diffraction (XRD), transmission
electron microscopy (TEM), fourier transform infrared spectroscopy (FTIR), and vibrating-sample
magnetometer (VSM) methods for purification and immobilization of the histidine-tagged recombi-
nant benzoylformate decarboxylase (BFD). The MSS support was shown to be eligible for selective
binding of HIS-tagged BFD by SDS-page analysis. Loading capacity of the MSS support was
determined as 43.6ꢀ 1.1 mg/g. The regeneration ability for protein binding was also studied. An
immobilized BFD was tested to catalyze benzoin condensation and representative cross acyloin
reaction. Conversion and enantiomeric excess values were comparable with that of free enzyme
catalyzed reactions. Chirality 00:000-000, 2013. © 2013 Wiley Periodicals, Inc.
KEY WORDS: metal-affinity magnetic nanoparticles; his-tagged protein purification;
benzoylformate decarboxylase (BFD); carboligation reactions
INTRODUCTION
purposes, the enzyme also accepts aldehydes as donors,
instead of R-ketoacids, to give the corresponding acyloins with
(S)-configuration.16 BFD which has been characterized and
cloned to an overexpressing E. coli with a histidine tag
for immobilized metal affinity chromatography (IMAC)
purification17 is suitable for new immobilization applications
where hexahistidine-tag (6xHis-tag) interaction with the vacant
coordination positions of nickel or copper chelates can easily
take place. Benzaldehyde lyase (BAL), another thiamine pyro-
phosphate (TPP) depending carboligating enzyme has been
reported for immobilization on metal coordinated surface mod-
ified particles for batch and continuous applications.8,18–21 To
date BFD has not been reported in metal binding purifica-
tion/immobilization applications. A few studies have been
performed on the immobilization of BFD to increase the cost
effectiveness of the reactions for continuous processes,22 and
to stabilize the enzyme in order to be used in organic media.23
In this study, a heterogeneous biocatalyst offering the ease
of immobilization along with the separation steps of metal
affinity ligand (Cu2+-IDA) coated magnetic nanoparticles is
introduced. The surface modified magnetic system reported
herein is also employed in the purification of crude BFD for
free enzyme catalyzed carboligation reactions. For prepara-
tion, first magnetic Fe3O4@SiO2-IDA-Cu2+ particles were
synthesized by the sol–gel method via surface modification.
The condensation products of 3-glycidoxypropyltrimethoxy
silane (GPTMS) and iminodiacetic acid (IDA) were anchored
onto magnetic silica nanospheres as ligands for the Cu2+ com-
plexes. The product was then charged with copper ions and
applied to the immobilization of His-tagged BFD. A magneti-
cally responsive nanobiocatalyst system for purification of
Magnetic nanoparticles (microspheres, nanospheres and
ferrofluids) have been studied extensively due to their unique
magnetic properties and potential applications in the frontier
fields such as data storage technology, environmental and
biomolecular separation, magnetic resonance imaging, drug
delivery, enzyme immobilization and immunoassay applica-
tions.1–5 In general, nanoparticles require a support for
further application. Since silica can protect the particles from
intersystem dipolar interactions, it has been exploited as a
coating material for magnetic nanoparticles. Silica surfaces
are often terminated by silanol groups that can react with
organosilane coupling agents to immobilize specific ligands.
This capability allows magnetic carriers to be used for nucleic
acid separation,6 enzyme immobilization,7,8 and hyperthermia
to name a few.9
Numerous works have been reported on the application of
metal ion-immobilized silica10,11 and magnetic silica supports12,13
in protein separation due to the stability of the metal chelates
formed and high protein loading capacity. Furthermore, metal
ion-immobilized and core/shell-structured magnetic/silica
composites have generated much interest because they can
be subject to simple magnetic separation and selective metal-
affinity purification. When an oligopeptide of six or seven
histidine residues is linked to the N- or C-terminus of an
enzyme, the resulting His-tagged enzyme can be strongly
immobilized onto the support material through metal chelates,
due to the cooperative bonding of the histidines to a metal ion
such as Cu2+.8,13
Benzoylformate decarboxylase (BFD) from Pseudomonas
putida whose native function is to decarboxylase benzoylformate
to benzaldehyde in the mandalate pathway has been widely
investigated for its potential use as a biocatalyst. Wilcocks and
Ward reported the carboligase activity of BFD from P. putida
for the first time, introducing the catalyst as an efficient
alternative for the enantioselective synthesis of (S)-2-
hydroxyketones.14 Its stereoselectivity is highly dependent on
the structure of the substrate aldehydes.15 For synthetic
Contract grant sponsor: Scientific and Technological Research Council of
Turkey; Contract grant number: TUBITAK, 109 T497.
*Correspondence to: Bilsen Tural, Department of Chemistry, Dicle University,
21280, Diyarbakir, Turkey. E-mail: bilsentural@gmail.com
Received for publication 7 January 2013; Accepted 22 March 2013
DOI: 10.1002/chir.22181
Published online in Wiley Online Library
(wileyonlinelibrary.com).
© 2013 Wiley Periodicals, Inc.

TURAL ET AL.
1 mL of TEOS were added consecutively. Reaction was allowed to proceed
BFD by affinity adsorption from crude cell extract and appli-
cation of this system for the carboligation reactions to form
(R)-benzoin (3) starting from benzaldehyde (1) and (S)-2-
hydroxy-l-phenyl-propanone (HPP) (4) starting from benzalde-
hyde (1) and acetaldehyde (2) are presented (Scheme 1).
at room temperature for 12h under continuous stirring. The resultant prod-
uct was obtained by permanent magnet separation and thoroughly washed
with deionized water six times (10 mL each time).
Preparation of IDA-Cu2+ Modified Fe3O4@SiO2 Magnetic
Nanoparticles
EXPERIMENTAL METHODS
The process for synthesis of IDA-Cu2+ modified Fe3O4@SiO2 magnetic
nanoparticles is shown in Scheme 2. In a typical recipe,13 IDA (4.25 g)
was dissolved in 50 mL deionized water. The pH was adjusted to 11.0 with
10 M NaOH. Then the solution was transferred to a flask placed in an ice-
bath at 0 ꢂC, and 1.4 mL of GPTMS was slowly added under stirring. The
mixture was heated up to 65 ꢂC and kept 6 h under stirring, subsequently
placed into an ice-bath for 5 min to decrease the temperature to 0 ꢂC,
again followed by the addition of 1.6 mL of GPTMS. Then, the solution
temperature was raised to 65 ꢂC where it was mixed for another 6 h.
Finally, the pH of the GPTMS-IDA solution was adjusted to 6 with concen-
trated HCl. 1 g of Fe3O4@SiO2 nanoparticles were added and the suspen-
sion was mixed at 95 ꢂC for 2 h. The final nanoparticles denoted as
magnetic Fe3O4@SiO2-IDA were separated with the help of permanent
magnet and washed thoroughly with deionized water (six times, 10 mL
each time).
Materials
All chemicals used were of analytical grade, commercially available and
used without any purification. Recombinant E.coli SG13009 strain
carrying the histidine tagged BFD gene was a kind gift of Dr. Martina
Pohl from CLIB-Graduate Cluster Industrial Biotechnology, Germany.
Chemicals used in production of BFD such as isopropyl-b-D-
galactopyranoside (IPTG) and antibiotics were all of molecular biology
grade and obtained from AppliChem (Darmstadt, Germany).
Characterization Techniques
The crystal structure of the Fe3O4@SiO2 nanoparticles was identified
using a diffractometer (Rigaku MiniFlex X-Ray diffractometer/PW 3710,
Tokyo, Japan) equipped with Cu-Ka radiation. The crystal size of iron
oxide particle in the iron oxide-silica nanocomposite was estimated using
the X-ray line broadening technique based on Scherer’s formula.
In charging the Fe3O4@SiO2-IDA magnetic nanoparticles with Cu2+
ions, first 30 mg of Fe3O4@SiO2-IDA magnetic nanoparticles were mixed
with 50 mL of aqueous CuSO4 (5 mg/mL) solution at 100 rpm for 1 h to
attain equilibrium. Then unbounded Cu2+ ions were removed with water.
The schematic illustration for the preparation steps of Cu+2 charged
Fe3O4@SiO2-IDA nanoparticles used for BFD adsorption is given in
Scheme 2C. Cu+2 charged Fe3O4@SiO2-IDA nanoparticles were named
magnetic solid support (MSS).
Fe3O4@SiO2 magnetic nanoparticles were redispersed in pure water by
sonication for 10 s and a drop of suspension was placed onto SPI Double
Copper Grids 100/200. The particles were detected by transmission
electron microscopy (TEM) (JEOL 2100 F, Tokyo, Japan) for particle size
and morphology. Fourier transformed infrared (FT-IR) spectra were mea-
sured on a Thermo Scientific Nicolet IS10 FT-IR spectrometer (Waltham,
USA). Sixteen scans were collected at a resolution of 4 cmꢁ1. The particle
sizes and distributions of Fe3O4@SiO2 magnetic nanoparticles were de-
termined by using Dynamic Light Scattering Technique (Zeta
SizerNano-ZS, Malvern). The measurement temperature was kept at
25 ꢂC. A vibrating-sample magnetometer (VSM, Q-3398, Cryogenic,
London, England) was used at room temperature to study the magnetic
properties of Fe3O4@SiO2 magnetic particles.
BFD-catalyzed reactions were monitored by thin layer chromatography
(TLC) on silica gel (Merck, Darmstadt, Germany). Spots were detected
by both UV-absorption and phosphomolybdicacid (PMA) staining. The
products were identified by 1H-NMR and 13C-NMR spectra (BRUKER
DPX 400 MHz) by using tetramethylsilane (TMS) as an internal standard
and deutero-chloroform as the solvent. Enantiomeric excess values were
determined via HPLC analysis (Agilent 1100 series, Chiralpak AD).
Purification of BFD by Using Magnetic Fe3O4@SiO2-IDA-
Cu+2 Nanoparticles
Recombinant E. coli SG13009 strain carrying pKK233-2 plasmid with
histidine tagged BFD gene was used in this study. Cells were grown in
100 mL LB (luria broth) medium until the optical density reached 0.5 at
600 nm, and 1 mM isopropyl-b-D-thiogalactopyranosid (IPTG) was added
for induction. After 6 h, cells were collected by centrifugation and
sonicated in 10mL 50mM potassium phosphate buffer (pH: 7.8, 10mM
NaCl and 20 mM imidazole) for crude cell lysate preparation. After centrifu-
gation, 1 mL of supernatant was used in purification with 100mg MSS (the
Fe3O4@SiO2-IDA-Cu+2 nanoparticles). First the lyophilized MSS was
washed twice with 3 mL phosphate buffer (with the same composition used
in extraction). To this equilibrated MSS, 2 mL buffer and 1 mL crude cell
lysate were added and incubated at 4ꢂC for 20 min under gentle shaking.
MSS was then separated from the slurry by applying a magnetic field
created by the permanent magnet and then washed with buffer twice.
MSS was separated and eluted with 3 mL buffer containing 200 mM imidaz-
ole. The eluted sample was then applied to sephadex G-25 (SIGMA)
desalting column for exchanging the buffer to reaction buffer concentra-
tions used for free enzyme reactions. For experiments performed with
immobilized system after adsorption of the enzyme, the slurry was washed
twice with 3 mL phosphate buffer and then once with 3 mL reaction buffer.
The schematic illustration for the BFD adsorption is given in Scheme 2C.
Protein amount in crude cell extract and purified enzyme solutions was
determined by the Bradford method using BSA (Bovine serum albumin)
standard protein assay.25 For SDS-PAGE gel analysis, 35 mL eluted protein
solution was applied to a 12.5% SDS-PAGE gel and color development was
performed by silver staining method.26
Synthesis of Fe3O4@SiO2 Magnetic Nanoparticles
Magnetite nanoparticles were prepared by the chemical coprecipitation
method.13 A complete precipitation of Fe3O4 (1 g) was achieved under
alkaline conditions, while maintaining a molar ratio of Fe2+: Fe3+ = 1:2
(0.86 g FeCl2.4H2O: 2.36 g FeCl3.6H2O) under nitrogen gas to prevent
critical oxidation in 40 mL of deaerated deionized water with vigorous
stirring (1000 rpm). As the solution was heated up to 80 ꢂC, 5 mL of
ammonium hydroxide was added. After 30 min, the resulting magnetite
nanoparticles were obtained by putting the vessel on an Nd–Fe–B perma-
nent magnet (maximum field strength of 4000 Oe) and decanting the
supernatant. The nanoparticles were washed with deionized water six
times (10 mL each time) to remove the unreacted chemicals.
The Fe3O4 nanoparticles were coated with silica using the sol–gel
method13,24 by dispersing 30mg of superparamagnetic Fe3O4 nanoparticles
in 80mL of 2-propanol and 6 mL of deionized water via sonication for approx-
imately 10 min. Then, to this mixture 7 mL of ammonium hydroxide and
Scheme 1. Carboligation reactions catalyzed by immobilized BFD.
Chirality DOI 10.1002/chir

CARBOLIGATION REACTIONS MEDIATED BY IMMOBILIZED BENZOYLFORMATE DECARBOXYLASE
Scheme 2. Schemes for A) Synthesis of GPTMS-IDA and B) Synthesis of Magnetic solid support, MSS C) Purification of BFD by using magnetic solid support.
To assess reusability for the purification of BFD, MSS was regenerated
five times by reloading with the same amount of crude cell lysate. BFD
loaded MSS was separated and eluted each time with 3 mL buffer
containing 200 mM imidazole. Protein amount in the purified enzyme
solutions was determined by Bradford method using BSA (bovine serum
albumin) standard protein assay.25
purification of the crude product by crystallization afforded
(R)-2-hydroxy-1,2-diphenylethan-1-one as a colorless solid;
yield: 82% (131 mg, 96% ee); mp 135 ꢂC [Lit.16 mp 133-134 ꢂC
22
D
for (R)-enantiomer]; ½aꢃ
¼
–112.1 (c 1.5, CH3COCH3);
22
[Lit.16 ½aꢃ
¼ –113.8 (c 1.5, CH3COCH3). HPLC (Chiralpak
D
Carboligation Reactions
AD) Rt(R ):27.1 min. Rt(S ):34.5 min. dH(400 MHz; CDCl3/
CCl4; Me4Si) 7.8 (2H, d, J = 7.8 Hz, Ph), 7.4 (1H, t, J = 7.5 Hz,
Ph), 7.3 (2H, t, J = 7.6 Hz, Ph), 7.1-7.2 (5H, m, Ph), 5.7 (1H, d,
J = 5.9 Hz, CH), 4.42 (1H, J = 5.9 Hz,, OH). dC(100 MHz;
CDCl3/CCl4; Me4Si) 198.7, 139.1, 133.8, 133.6, 129.1, 129.0,
128.6, 128.5, 127.7, 76.2.
Reactions were performed both with immobilized enzyme on magnetic
solid support and free enzyme purified with the same amount of magnetic
solid support to compare the yield and ee values.
Synthesis of (R)-benzoin ((R)-2-hydroxy-1,2-diphenylethan-1-
one). Benzoin formation reaction with free enzyme was car-
ried out as described in the literature.16 Benzaldehyde
(212 mg, 2 mmol) was dissolved in a mixture of dimethyl sulfox-
ide (DMSO) (4mL) and potassium phosphate buffer [16 mL,
50mM, pH 7.0, containing MgSO4 (2.5mM) and ThDP
(0.15 mM)]. After addition of 350 mg immobilized BFD (3.5
U)27 the reaction mixture was allowed to stand at 30ꢂC for
120 min (the reaction is monitored by TLC and HPLC). The
reaction mixture was extracted with dichloromethane (50 mL)
and the organic layer washed with water (5 mL) and brine
(5mL) and dried over Na2SO4. Evaporation of the solvent and
Representative example for the cross acyloin reaction: Synthesis
of (S)-2-hydroxy-1-phenylpropanone [(S)-2-HPP].28 Benzalde-
hyde (106 mg, 1 mmol) was dissolved in a mixture of DMSO
(4 mL) and potassium phosphate buffer (16 mL, 50 mM,
pH 7.0, containing MgSO4 (2.5 mM) and ThDP (0.15 mM)).
To this solution 44 mg (1 mmol) acetaldehyde was added.
After addition of 200 mg immobilized BFD (58 U),27 the reac-
tion mixture was kept at 30ꢂC. Additional acetaldehyde
(88 mg-2 mmol) was introduced to the reaction medium on
Chirality DOI 10.1002/chir

TURAL ET AL.
hourly basis till conversion reached 97% (180 h). Work-up
(300ꢂK) (shown in Fig. 2). The magnetization curve exhibits
zero remanence and coercivity, which proves that these silica
nanoparticles have superparamagnetic properties. The
superparamagnetism enables the nanoparticles to respond to
an applied magnetic field without any permanent magnetization
and redisperse rapidly when the magnetic field is removed.
The saturation magnetization of the obtained magnetic
Fe2O3@SiO2 nanoparticles is 16 emu/g. This large saturation
magnetization of magnetic silica nanoparticles makes them
very susceptible to magnetic fields, and therefore makes the
solid and liquid phases separate easily.13
according to the former procedure28 afforded 132 mg (88%).
22
D
Viscous oil, ee > 92%; ½aꢃ
¼ -85.1 (c 2.0, CHCl3), HPLC
(Chiralpak AD): ) Rt(R): 14.7 min, Rt (S) : 12.5 min; dH
(400 MHz; CDCl3/CCl4; Me4Si) 7.9 (2H, m, Ph), 7.40-7.60
(3H, m, Ph), 5.1 (1H, q, J = 6.0 Hz, CH), 3.8 (1H, br s, OH).
dC(100 MHz; CDCl3/CCl4; Me4Si) 201.7, 134.3, 134.0, 128.9,
128.7, 69.2, 22.0.
Reusability of Immobilized BFD for Carboligation Reactions
The image in Figure 3 shows the TEM bright field
micrograph for the Fe3O4@SiO2 magnetic nanoparticles.
The average size of the nanoparticles was approximately
11 nm. The size determined by XRD using the Scherrer
equation correlated well with the actual size of the core–shell
magnetic nanoparticles.
MSS was characterized by FT-IR. In Figure 4, the peak at
around ~3090 cmꢁ1 assigned to C–H stretching vibration from
the methylene (-CH2-) in the silane coupling agent and absorp-
tion bands at ~1600 and ~1400 cm–1, assigned to the carboxylate
revealed that IDA molecules were successfully immobilized on
the surface of the magnetic silica nanoparticles.31,32
To assess the reusability of immobilized BFD for carboligation
processes, benzoin condensation reaction was carried out for five consec-
utive batches with the same immobilized enzyme. For each period, the
immobilized BFD was separated with the help of a magnet, washed two
times with potassium phosphate buffer at pH 7.5 containing 50% DMSO
and incubated with fresh substrate for the next reaction. The residual
activity of the immobilized BFD was measured in terms of (R)-benzoin
concentration by HPLC after each reaction.
RESULTS AND DISCUSSION
Characterization
The crystal structure of Fe3O4@SiO2 magnetic nanoparticles
was investigated by XRD analysis. The XRD pattern is shown in
Fig. 1. The main peaks occur at 2 = 30.16ꢂ, 35.48ꢂ, 42.99ꢂ,
52.90ꢂ, 56.95ꢂ, and 62.70ꢂ, which correspond to (220), (311),
(400), (422), (511), and (440), respectively. The diffraction
pattern of the magnetic silica nanoparticles obtained within this
study was close to the standard pattern for crystalline magne-
tite (Fe3O4) or maghemite (g-Fe2O3) with an inverse spinel
structure.29 However, because of very similar magnetic proper-
ties, it was not easy to distinguish between these two types. It is
reasonable to assume that the magnetic silica nanoparticles of
concern would be a mixture of Fe3O4 and g-Fe2O3 because of
the atmospheric conditions during preparation and handling
procedures which would result in the change of some Fe3O4
to g-Fe2O3. The average particle size of the magnetite
nanoparticles coated with silica was estimated to be 11.8 nm
by using the Scherrer equation30 (d = 0.9l/(2)cos, where d is
the crystalline domain size, (2) is the width at half maximum
of the strongest peak (311), and l is the x-ray wavelength).
To study the magnetic properties of magnetite nanoparticles
after silica coating, we registered the plots of the magnetization
M versus the applied magnetic field H at room temperature
The particle size distribution of Fe3O4@SiO2 magnetic
nanoparticles is shown in Figure 5. According to Figure 5
the average particle agglomeration size of Fe3O4@SiO2
particles is ~500 nm.
Purification of BFD
First, MSS was employed for one-pot purification-
immobilization. From the band corresponding to the molecu-
lar weight 56 kDa of BFD33 (Fig. 6) MSS developed in this
study was shown to be effective for the selective binding of
HIS-tagged BFD.
The adsorption capacity of the Cu+2 charged magnetic solid
support for histidine tagged BFD was determined by
standard BSA assay as 43.6 ꢀ 1.1 mg BFD/g-magnetic solid
support. Highest protein elution was obtained with 200 mM
imidazole. When higher imidazole concentrations were
applied for elution of the adsorbed protein, protein concentra-
tions were not changed. The MSS was regenerated five times
by reloading with the same amount of crude cell lysate. The
Fig. 2. Magnetization curve of Fe3O4@SiO2 magnetic nanoparticles
obtained by VSM at room temperature.
Fig. 1. X-ray diffraction pattern of Fe3O4@SiO2 magnetic nanoparticles.
Chirality DOI 10.1002/chir

CARBOLIGATION REACTIONS MEDIATED BY IMMOBILIZED BENZOYLFORMATE DECARBOXYLASE
Fig. 6. SDS-PAGE analysis of the purified 6Xhis tagged BFD. M, SDS-PAGE
Fig. 3. TEM graphs of Fe3O4@SiO2 magnetic nanoparticles.
molecular weight marker; 1, crude extract of E.coli BL21(DE3)pLysS containing
BFDHIS; 2–4, protein eluted from 100, 700, 1500 μl crude extract loaded mag-
netic solid support.
Fig. 7. Protein binding capacity of MSS for repeated number of experiments.
Fig. 4. FT-IR spectra of: (A) IDA-functionalized Fe3O4@SiO2 magnetic
nanoparticles (B) pure IDA (C) Fe3O4@SiO2 magnetic nanoparticles.
Carboligation Reactions
Benzoin condensation reaction. The benzoin condensation
reaction of immobilized BFD was carried out as previously
reported by Demir17 and kinetic analysis was performed by
following the increase in the benzoin concentration (Fig. 8).
Benzaldehyde was employed at a weight interval of 53 mg to
318 mg. At the 120th min, 212 mg benzaldehyde resulted in
an 82% conversion and a decrease in the benzoin concentra-
tion occurred at subsequent time points. When the reactions
were carried for a prolonged time, in all of the cases including
a free enzyme reaction, product inhibition occurred (apparent
from the declines in the reaction curves). and at higher
substrate concentration (424 mg), no conversion was detected.
When 318 mg of benzaldehyde was applied, we observed that
the enzyme activities decreased to the lowest level due to the
enzyme inactivation and inhibition caused by the reaction product.
A similar observation was also reported by Drager et al. 8 and
Sopaci et al.34 with benzaldehyde concentration during the
benzaldehydelyase (BAL, EC 4.1.2.38 which is the same
class of enzyme) immobilized batch reaction and this
phenomenon was also explained by product inhibition of
ligase reaction. The activity of immobilized BFD under opti-
mized conditions was found to be comparable to that of free
enzyme and enzyme immobilized to magnetic solid support
Chirality DOI 10.1002/chir
Fig. 5. Particle size distribution of Fe3O4@SiO2 magnetic nanoparticles.
adsorption capacity was increased at the second adsorption,
achieved maximum value at third and dropped at the fourth
and fifth, still having a correlatively higher value than the first
load. This phenomenon can be explained by the increase in
surface to volume ratio of the silica coated particles in the
buffer during the first experiment period (Fig. 7).

TURAL ET AL.
Fig. 11. Response of the BFD-Magnetic solid support to the magnetic field.
Fig. 8. Course of benzoin formation with free enzyme (eluted from 350 mg
magnetic solid support) and immobilized BFD (on 350 mg magnetic solid sup-
port) at different benzaldehyde concentrations. 1: 212 mg (with free enzyme);
2: 212 mg, 3: 106 mg , 4: 53 mg and 5: 318 mg, (nr.2, nr.3, nr.4 and nr.5 with
immobilized enzyme).
Cross
acyloin
reaction:
Synthesis
of
(S)-2-hydroxy-1-
phenylpropanone [(S)-2-HPP]. BFD is also effective in cross
acyloin reaction of benzaldehyde with acetaldehyde to form
(S)-2-HPP. As reported earlier in the literature, the benzalde-
hyde/acetaldehyde ratio is very important for the product
distribution.35 Equal amounts of donor and acceptor aldehyde
furnished a mixture of (R)-benzoin and (S)-HPP but a low
concentration of benzaldehyde in the presence of excess
acetaldehyde resulting in the formation of (S)-2-HPP in 96%
ee and 84% yield.35
Carboligation reactions were performed for the formation of
cross acyloin product HPP with free and immobilized BFD to
find the effect of immobilization on enzyme activity. Reactions
were carried out until the desired conversions were achieved.
These reactions were screened with different amounts of
immobilized BFD to obtain maximum product formation (the
reaction was monitored by HPLC, see Fig. 10). The activity of
the immobilized enzyme was slightly higher due to the stability
caused by immobilization while in the reactions with free
enzyme, enzyme activity decreased dramatically most probably
due to lyophilization during the purification. In both cases, the
ee values of the products were >92%.
Fig. 9. Remaining activity of the immobilized BFD with a repeated set of
experiments.
Since the enzyme-magnetic solid support was highly
responsive to the magnetic field (Fig. 11), samples were
withdrawn after the slurry of the carboligation reactions
which were clarified by the application of an external magnet.
CONCLUSIONS
Magnetic Fe3O4@SiO2 nanoparticles were prepared using
the sol-gel technique and their surfaces were modified to
obtain a functional product possessing high affinity for BFD.
First, GPTMS-IDA was introduced onto the surfaces of
Fe3O4@SiO2 nanocomposite particles, and subsequently Cu2+
ions were attached to them. The magnetic support provided
high adsorption capacity for BFD (43.6ꢀ 1.1 mg/g magnetic
solid support). When the magnetic solid support was
regenerated five times, its BFD adsorption capacity did not
change considerably. Finally, it has been shown that the purifi-
cation of BFD using the magnetic support based on magnetic
separation is a relatively easy and cost effective procedure.
Since this magnetic support is easily dispersive in medium it
is convenient and effective for protein binding and elution
procedures. In the same respect this method is also found to
be promising for one pot purification and immobilization proce-
dures for histidine tagged recombinant enzymes. BFD was
efficiently immobilized on the magnetic particles and used as
Fig. 10. Synthesis of 2-HPP with various benzaldehyde concentrations, 1:
With free enzyme (eluted from 100 mg magnetic solid support, reaction
performed with 100 mg benzaldehyde mL-1), 2: With immobilized enzyme
(BFD immobilized on 100 mg magnetic solid support) 50mMKpi, pH 7.0, 2
mM MgSO4, 0.1 mM ThDP, 500 mM acetaldehyde, 30 ꢂC, 24 h).
containing an equal amount of protein with nanoparticles.
The enantiomeric exces of benzoin was obtained as 96%.
Experiments were repeated with the same batch of
immobilized BFD after washing with 50% DMSO in a lysis
buffer. The enzyme was shown to be effective without any
loss of activity for the three sets of experiments and the
remaining activity decreased during the fourth and fifth
repetition (90% and 83% respectively) (Fig. 9).
Chirality DOI 10.1002/chir

CARBOLIGATION REACTIONS MEDIATED BY IMMOBILIZED BENZOYLFORMATE DECARBOXYLASE
15. Knoll M, Müller M, Pleiss J, Pohl M. Factors mediating activity, selectivity,
a heterogeneous catalyst for representative BFD mediated
carboligation reactions. These magnetically responsive
particles can be used for the direct immobilization and easy
separation of enzymes from a crude extract in order to obtain
pure immobilized enzyme as well as a direct use for catalytical
purposes. In addition, two important representative reactions
were performed with this system in which the products were
obtained in high chemical and optical yields. Additionally, the
reusability of the immobilized enzyme (minimum five times
without losing considerable activity) is another advantage
compared to free enzymes. To sum up, a new heterocatalyst
system for BFD mediated acyloin reactions via C-C bond forma-
tion reactions with a high optical and chemical yield (ee > 96%)
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This project was funded by the financial support from Middle East
Technical University, Dicle University Research Fund (DUBAP,
Project No.10-ZEF-28, Project No. 13-ZEF-28 and Project No. 12-
ZEF-25) and the Scientific and Technological Research Council
of Turkey (TUBITAK, 109 T497), COST Action CM0701.
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Chirality DOI 10.1002/chir