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B. Tural et al. / Journal of Molecular Catalysis B: Enzymatic 95 (2013) 41–47
bilization.
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, Japan) for
particle size and morphology.
On the other hand, one of the most suitable methods for
industrial-scale immobilization of proteins is based on epoxy
supports [18–20]. Epoxy supports present many advantages; for
example, they are very stable, allowing for long-term storage, pro-
longed transport from manufacturer to consumer, and extended
enzyme-support reaction periods. In addition, they are reactive
with different moieties of proteins (amine, thiol, hydroxyl groups),
yielding very stable protein-support bonds (secondary amine,
easily blocked after enzyme immobilization with different com-
pounds, yielding an inert surface.
The mechanism of enzyme immobilization in epoxy supports
to purification. In fact, it has been described that adsorption of
the protein on the epoxy resin is necessary to obtain a significant
covalent immobilization because of the extremely low reactivity
of the epoxy supports with soluble proteins [21–23]. Therefore,
due to the low reactivity of the epoxy groups, immobilization of
proteins on epoxy supports follows a two step mechanism: (1)
the enzyme is hydrophobically adsorbed on a fairly hydrophobic
support at very high ionic strength; and (2) the covalent bond-
ing between the enzyme and the support proceeds. Using this
premise, the use of multifunctional epoxy supports to immobilize
proteins has been reported [24]. These epoxy supports have differ-
ent moieties that are able to physically adsorb proteins via different
structural features, plus a dense layer of epoxy groups able to cova-
lently bind the enzyme. One of the multifunctional supports that
may be easily produced is the metal chelate epoxy support [24–26].
These supports combine the good properties of epoxy supports for
enzyme immobilization and stabilization with an increased pos-
sibility for IMAC chromatography for purification poly-His-tagged
proteins.
In this study, chelate-epoxy modified SCMPs nanoparticles are
optimized to achieve the one step purification, covalent immobi-
lization, and stabilization via multipoint covalent attachment of
6XHis-tagged BAL. The chelate-epoxy modified SCMPs (the sil-
ica coated magnetite particles) give high yields during acyloin
condensation reactions through the covalent immobilization of
the BAL on a magnetically responsive epoxy-chelate support sys-
tem. The epoxy-chelate modified SCMPs were fully characterized.
Experimental conditions for surface modification, protein immobi-
lization and purification were investigated and optimized. The BAL
immobilized chelate-epoxy modified SCMPs were applied for the
ligase activity of the BAL during the self condensation of benzalde-
hyde.
Fourier transformed infrared (FT-IR) spectra were measured on
a Thermo Scientific Nicolet IS10 FT-IR spectrometer (USA). Sixteen
scans were collected at a resolution of 4 cm−1
.
The particle sizes and distributions of the SCMPs were
determined by using Dynamic Light Scattering Technique (Zeta
SizerNano-ZS, Malvern). The measurement temperature was kept
at 25 ◦C.
The BAL-catalyzed reaction was monitored by thin layer chro-
matography (TLC) on silica gel (E. Merck, Darmstad). The detection
of spots was performed by both UV-absorption and phospho-
molybdicacid (PMA). The product synthesized was identified by
1H NMR and the 13C NMR spectra were recorded by BRUKER DPX
400 MHz by using tetramethylsilane (TMS) as an internal standard
and deutero-chloroform as a solvent. The reaction was followed by
2.3. Synthesis of epoxy-functionalized SCMPs
Magnetite nanoparticles were prepared by the chemical copre-
cipitation method [29]. A complete precipitation of Fe3O4 was
achieved under alkaline conditions, while maintaining a molar
ratio of Fe2+:Fe3+ = 1:2 under a nitrogen gas environment to
prevent critical oxidation. To obtain 1 g of Fe3O4 nanoparticles,
0.86 g of FeCl2·4H2O and 2.36 g of FeCl3·6H2O were dissolved
under a N2 atmosphere in 40 mL of deaerated deionized water
with vigorous stirring (1000 rpm). As the solution was being
heated to 80 ◦C, 5 mL of ammonium hydroxide was added. After
30 min, the resulting magnetite nanoparticles were obtained by
putting the vessel on a permanent magnet and the supernatant
water six times (50 mL each time) to remove unreacted chemi-
cals.
The Fe3O4 nanoparticles were coated with silica using the
sol–gel method [29]. Typically, 30 mg of superparamagnetic Fe3O4
nanoparticles was dispersed in 80 mL of 2-propanol and 6 mL of
deionized water by sonication for about 10 min. Then, under contin-
uous mechanical stirring, 7 mL of ammonium hydroxide and 1 mL of
TEOS were consecutively added to the reaction mixture. The reac-
tion was allowed to proceed at room temperature for 12 h under
continuous stirring. The resultant product was obtained by mag-
netic separation with the help of the permanent magnet and was
thoroughly washed with deionized water six times (50 mL each
time).
One gram of wet SCMPs was reacted with 10 mL of 5% GPTMS in
toluene at room temperature overnight. After the coupling reaction,
the modified magnetic nanoparticles were removed from the solu-
tion with the help of the permanent magnet and rinsed thoroughly
Finally, they were freeze-dried. When prepared in this way, the
groups.
The schematic illustration for the preparation steps of the
epoxy-functionalized SCMPs is shown in Scheme 1. The epoxy con-
tent of the SCMPs was determined using the pyridine–HCl method
given in the literature [30]. This analysis indicated that 0.30 mmol
epoxy groups were attached to per gram of SCMPs.
2. Materials and methods
2.1. Chemicals and materials
(DE3) PLysS strain (host to produce the recombinant BALHIS) was
purchased from Invitrogen®. Plasmid was a generous gift from Dr.
Martina Pohl (Institute of Biotechnology 2, Research center Jülich,
Germany) [2,27,28]. Enzyme production was performed in a New
Brunswick BioFlo110 Fermenter equipped with pH and tempera-
ture probes as well stirring rate controls.
2.4. Preparation of IDA-epoxy-functionalized SCMPs
2.2. Characterization
Epoxy functionalized SCMPs (1 g) was incubated in 15 mL of
0.1 M sodium borate/2 M iminodiacetic acid (pH 8.0) at room
temperature overnight under very gentle stirring. The support
Silica coated magnetic nanoparticles were re-dispersed in pure
water by sonication for 10 s. The particles were re-dispersed in pure