Activity of Candida rugosa lipase immobilized on γ-Fe2O3 magnetic nanoparticles

Article abstract of DOI:10.1021/ja021223n

We report the stability and enzymatic activity of Candida rugosa Lipase (E.C.3.1.1.3) immobilized on γ-Fe₂O₃ magnetic nanoparticles. The immobilization strategies were either reacting the enzyme amine group with a nanoparticle surfac

Full text of DOI:10.1021/ja021223n

Published on Web 01/28/2003
Activity of Candida rugosa Lipase Immobilized on γ-Fe2O3 Magnetic
Nanoparticles
Ansil Dyal,†,‡ Katja Loos,†,‡ Mayumi Noto,† Seung W. Chang,†,‡ Chiara Spagnoli,†
Kurikka V. P. M. Shafi,†,‡ Abraham Ulman,*,†,‡ Mary Cowman,† and Richard A. Gross†
Department of Chemical Engineering, Chemistry and Material Science, Polytechnic UniVersity, 6 Metrotech Center,
Brooklyn, New York 11201, and The NSF Garcia MRSEC for Polymers at Engineered Interfaces
Received September 30, 2002 ; E-mail: aulman@duke.poly.edu or aulman@photon.poly.edu
The use of nanophase materials offers many advantages due to
their unique size and physical properties. Hybrid nanoscale materials
are well established in various bioprocesses such as nucleic acid
detachment,1 protein separation,2 and immobilization of enzymes.3
An important area of interest is the immobilization of proteins
and enzymes on magnetic particles. Several magnetic particles3,4
and magnetic supports such as microspheres of various biomaterials5
encapsulating the magnetic particles, and copolymers5 with magnetic
particles have been used with good results. However, due to size
constraints (usually 75-100 µm), these microparticles cannot be
placed at specific locations that are relevant to cellular biochemical
processes. Preferably, such particles would possess a very low
magnetic hysteresis and high stability. The functionalized γ-Fe2O3
magnetic nanoparticles used as a support in this communication
possess all these traits. In addition, because of the inherent structure
and size of these particles, they are superparamagnetic which
addresses aggregation and flocculation concerns.6 Here, we report
the stability and enzymatic activity of Candida rugosa lipase
(E.C.3.1.1.3) immobilized on γ-Fe2O3 magnetic nanoparticles.
Lipases are frequently employed enzymes as they are commonly
Figure 1. The strategies used to immobilize Candida rugosa lipase on the
γ-Fe2O3 nanoparticles.
used for the synthesis of enantioenriched monomers and macromers
and for polymerization reactions.7 It is shown here that these
enzymes, when immobilized on magnetic γ-Fe2O3 nanoparticles,
microwave irradiation for 10 min. Nucleophilic substitution using
2-thiophene thiolate resulted in thiophene-functionalized nanopar-
ticles. These were either acetylated using acetic anhydride with
iodine as catalyst or reacted with nitrosonium tetrafluoroborate in
methylene chloride to produce the nitroso derivative. Given the
chemistry of thiophene, we assume that in both cases electrophilic
substitution occurred at the 5-position. In all cases full coverage
of the surface area was achieved which was proven by thermo-
gravimetrical analysis. Notice that coating of the γ-Fe2O3 with 11-
bromoundecanoic acid decreases the magnetization of the bare
γ-Fe2O3 on average only by 12%, resulting in nanoparticles with
value still far greater than any previously reported magnetic support.
The acetylated nanoparticles were reacted directly with the
enzyme, which was chemically bonded to the nanoparticle surface
via a CdN bond. The nitroso-functionalized nanoparticles were
reduced to the corresponding amine-functionalized nanoparticles
with SnCl2, and the enzyme was chemically connected using
glutaraldehyde.
For the immobilization on acetylated nanoparticles, 17.3 mg of
lipase (ca. 8% protein content according to protein assay) was
reacted with 32.38 mg of the acetylated nanoparticle in 8 mL of
phosphate buffer (10 mM, pH 7.5) under gentle shaking for 24 h
at room temperature. The immobilization on amino-functionalized
nanoparticles was conducted in a one- or two-pot reaction,
respectively. In the one-pot approach, 30 mg of amino-fuctionalized
nanoparticles was mixed with 5 µL of a 50 wt % solution of
glutaraldehyde in water and 2 mL of phosphate buffer and was
shaken for 1 h at 25 °C. Thereafter, 92 mg of lipase was added in
can be easily separated from the reaction medium, stored, and reused
with consistent results. This system offers a relatively simple tech-
nique for separating and reusing enzymes over a longer period than
that for free enzymes alone and for enzymes which are immobilized
by physisorption. This can be explained by the use of covalent im-
mobilization that does not permit the loss of enzyme by desorption
from the support and protects the enzyme from denaturation by
constraining it to the local environment of the particle.8 However,
while the ability to stabilize and recover the enzyme is achieved
by chemical immobilization, some enzymatic activity may be lost
by chemical bonding since the active site is hidden or restricted
from assuming the conformation needed to initiate the catalysis.9
For the immobilized enzyme in the present case separation is
facilitated by the use of a magnet where either the substrate solution
is removed while the immobilized enzyme is held in place with a
magnetic field or vice versa.
γ-Fe2O3 nanoparticles were prepared by sonication of Fe(CO)
in decalin and the subsequent annealing of the amorphous Fe2O3
nanoparticles.10 The average size of the γ-Fe2O3 nanoparticles is
20 ( 10 nm (see Figure 2), with saturation magnetization value of
61emu/g.10 Figure 1 presents the strategy used to immobilize
Candida rugosa lipase on the γ-Fe2O3 nanoparticles. 11-Bromoun-
decanoic acid was covalently linked to the nanoparticle surfaces
by heating the nanoparticles in acid solution in ethanol by using
† Polytechnic University.
‡ The NSF Garcia MRSEC for Polymers at Engineered Interfaces.
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J. AM. CHEM. SOC. 2003, 125, 1684-1685
10.1021/ja021223n CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. Enzymatic activity of Candida rugosa lipase immobilized on
γ-Fe2O3 nanoparticles as a function of time (days). (s0 1; - - -O 2; -× 3;
- - -× 4; - - -b 5; - - -4 8; sO 9; s2 10; - - -2 15; s∆ 16; sb 23; - - -[
24; - - -2 25; - - -[ 26; s[ 27).
Figure 2. AFM 3D height image of the enzyme magnetic nanoparticles
moieties. Average size is 20 ( 10 nm after deconvolution.11
The loss in activity is significantly higher than that reported for
enzymes immobilized on micrometer-sized polymeric beads using
physisorption. However, in that latter case it was observed in our
laboratory that the enzyme desorbs from the beads when they are
exposed to solution, suggesting that at least some of the reaction
was carried out in solution and not on the surface support. This is
not the case here, where the enzymes are chemically bonded to the
nanoparticles.
The most significant advantage of our samples is their long-
term stability. We have observed constant activity over one month
in the case of lipase immobilized on acetylated nanoparticles, as
can be seen in Figure 3. Our hybrid enzyme-nanoparticle
composites show just ∼15% decrease in activity over that period
probably due to desorption or denaturation (approximately 2%
reduction was noticed over a time period of 14 days for lipase
immobilized on amino-functionalized nanoparticles). These experi-
ments were carried out by separating the nanoparticles from the
solution at the end of each day, washing them with buffer solution,
introducing them to freshly prepared ester solutions, and measuring
the reaction kinetics. This (so-far unreported) long-term stability
illustrates the advantage of attaching the enzymes chemically to
the nanoparticles. Such stability might make economically viable
the use of expensive enzymes and hence opens a new horizon for
enzymatic catalysis in biotechnology.
4 mL of phosphate buffer and immobilized for 23 h at room
temperature. In the two-pot approach, 30 mg of amino-function-
alized nanoparticles was mixed with 50 µL of a 50 wt % solution
of glutaraldehyde in water and 2 mL of phosphate buffer and was
shaken for 3 h at 25 °C. The glutaraldehyde solution was removed
by decanting the solution out while holding the particles down with
a magnet, and the particles were washed three times with 4 mL of
phosphate buffer; 92 mg of lipase in 4 mL of phosphate buffer
was immobilized by gentle shaking at room temperature for 21 h.
In all three approaches the product was separated from the
supernatant by holding the particles down with a magnet, taking
the solution out, and thereafter washing four times with 4 mL of
phosphate buffer.
The amount of immobilized enzyme was obtained by standard
BCA protein assays of the original lipase solution, the supernatants,
and washing solutions after immobilization, respectively (in the case
of the glutaraldehyde linker the supernatant and the wash solutions
were dialyzed to prevent false results in the BCA assay). In the
case of acetylated nanoparticles we obtained a loading of 5.8 µg
of protein per mg of nanoparticles. For the one-pot reaction with
amino-functionalized particles we obtained 55.6 µg per mg of nano-
particles and for the two-pot reaction, 22 µg per mg of nanoparticles.
Samples for AFM were prepared by spreading a small amount
of the enzyme-functionalized magnetic nanoparticles on a thin layer
of glue deposited on a steel disk. Imaging was performed using a
Nanoscope Multimode Scanning Probe Microscope, DI, equipped
with an EV scanner operating in tapping mode. Nanoprobe SPM
etched silicon tips (TESP), were used for scanning. Imaging was
performed in soft tapping conditions (amplitude setpoint between
1.8 and 2.000 V). To measure the particle size, the broadening effect
due to the tip has been taken into account.11
Images show a preference for particles to self-associate, probably
due to magnetic attraction, the absence of a strong interaction with
the substrate, and the procedure of sample preparation (Figure 2).
It is not possible to recognize discrete single enzyme nanoparticle
moieties because of the aggregation between particles.
The enzymatic activity of the immobilized lipase was determined
by following the ester cleavage of p-nitrophenol butyrate (100 mM
solution) via UV spectroscopy (Molecular Devices SpectraMax Plus
384 spectrometer, at 450 nm and 25 °C). The enzymatic activity
for Candida rugosa lipase immobilized on γ-Fe2O3 magnetic
nanoparticles is lower than that for the free enzyme, but in all three
cases the enzymatic activity was approximately equivalent (ace-
tylated 1.1 × 10-7, one-pot reaction 7.8 × 10-8, two-pot reaction
1.6 × 10-7 mol/min per mg of protein, in comparison with free
enzyme 2.6 × 10-5 mol/min per mg of protein). Autohydrolysis
of the p-nitrophenolbutyrate and ester cleavage by magnetic γ-Fe2O3
nanoparticles without enzyme was not observed.
Acknowledgment. We thank Professor J. Zlatanova for the use
of the Atomic Force Microscope. K.L. thanks the AvH Foundation
for financial support. This work was funded by the NSF through
the MRSEC for Polymers at Engineered Interfaces, and the Center
for Biocatalysis and Bioprocessing of Macromolecules.
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