Improving the catalytic activity of formate dehydrogenase from Candida boidinii by using magnetic nanoparticles

Article abstract of DOI:10.1016/j.molcatb.2012.03.021

Formate dehydrogenase from Candida boidinii (FDH) was immobilized on three different magnetic supports: one composed by magnetite nanoparticles directly silanized with APTS (aminopropyltriethoxysilane), i.e. MagNP-APTS; the second one containing a silica gel coated magnetite core which was further silanized with APTS (MagNP@SiO₂-APTS), and the third one consisting of magnetite-APTS coated with Glyoxyl-Agarose (MagNP-Glyoxyl-Agarose). The catalytic activity of the three FDH systems was investigated as a function of pH and temperature. The silica gel coated nanoparticles provided the highest conversion rates; however, in terms of recycling, magnetite without the silica shell led to the most stable system. By using the enzyme tryptophan residues as internal fluorescence probes, the structure-activity behavior was investigated in the presence of the formate and NAD⁺ substrates, revealing a rather contrasting behavior in the three cases. Because of its peculiar behavior, a direct interaction of the magnetic nanoparticles with the catalytic sites seems to be implicated in the case of MagNP-APTS.

Full text of DOI:10.1016/j.molcatb.2012.03.021

Journal of Molecular Catalysis B: Enzymatic 84 (2012) 136–143
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Improving the catalytic activity of formate dehydrogenase from Candida boidinii
by using magnetic nanoparticles
Caterina G.C.M. Nettoa, Marcelo Nakamuraa, Leandro H. Andradeb, Henrique E. Tomaa,∗
a Supramolecular NanotechLab, Institute of Chemistry, University of São Paulo, São Paulo, SP, Brazil
b Laboratory of Fine Chemistry and Biocatalysis, Institute of Chemistry, University of São Paulo, São Paulo, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 6 April 2012
Formate dehydrogenase from Candida boidinii (FDH) was immobilized on three different magnetic
supports: one composed by magnetite nanoparticles directly silanized with APTS (aminopropyltri-
ethoxysilane), i.e. MagNP-APTS; the second one containing a silica gel coated magnetite core which was
further silanized with APTS (MagNP@SiO2-APTS), and the third one consisting of magnetite-APTS coated
with Glyoxyl-Agarose (MagNP-Glyoxyl-Agarose). The catalytic activity of the three FDH systems was
investigated as a function of pH and temperature. The silica gel coated nanoparticles provided the highest
conversion rates; however, in terms of recycling, magnetite without the silica shell led to the most stable
system. By using the enzyme tryptophan residues as internal fluorescence probes, the structure-activity
behavior was investigated in the presence of the formate and NAD+ substrates, revealing a rather con-
trasting behavior in the three cases. Because of its peculiar behavior, a direct interaction of the magnetic
nanoparticles with the catalytic sites seems to be implicated in the case of MagNP-APTS.
© 2012 Elsevier B.V. All rights reserved.
Keywords:
Formate dehydrogenase
Immobilization
Magnetic nanoparticles
1. Introduction
enzyme is its role as one of the catalysts in the enzymatic con-
version of CO2 to methanol [14–16], which is important for the
Immobilization has effectively been used as a method for
increasing the stability of enzymes and many reports have also
suggested the possibility of improving the enzyme activity and
selectivity [1,2]. Several material supports and methods have
already been studied, but in particular magnetic nano (or micro)
particles seem especially attracting since they can be easily recov-
ered by the use of an external magnet. In addition, the use of an
immobilized enzyme [3–5] introduces a great simplification on
the reactor design and control of the reactions, since the simple
removal of the enzyme effectively allows to stop the process. This
advantage is stimulating the use of magnetic nanoparticles in bio-
catalysis.
The conversion of CO2 to formate in biological systems is typ-
ically performed by a NADH dependent enzyme called formate
dehydrogenase (FDH), which is also used for catalytical studies
involving hydride transfer, due to the substrate simplicity. A typical
biotechnological use of FDH is in the determination of formic and
oxalic acid in solution, but it can also be used as a NAD+ regeneration
system [6–8], especially after immobilization, e.g. on silica matrixes
[9], agaroses [1,10], membranes [11] and magnetic nanoparticles
[12]. The best recyclability performance has been obtained using
alginate gels as supports [13]. Another important feature of this
development of new routes to fabrication of fuels and chemicals.
In spite of the previous studies on FDH immobilization in the
literature [17], a systematic study focusing on the improvement
of the enzyme activity and stability in immobilized systems is yet
missing.
Hence, we performed the immobilization of FDH from Candida
boidinii on three types of functionalized magnetite nanoparticles:
MagNP-APTS, MagNP@SiO2-APTS and MagNP-Glyoxyl-Agarose,
pursuing the understanding of the relevant parameters for improv-
ing the performance and recycling the FDH enzyme.
2. Experimental
All reagents were purchased from Sigma–Aldrich and used with-
out previous purification.
Formate
dehydrogenase
from
C.
boidinii
exhibited
5–15 units/mg of protein. One unit expresses the oxidation of
1 ␮mol of formate to CO2 in the presence of NAD+ at pH 7.6 and
37 ◦C.
2.1. Synthesis of MagNP, MagNP@SiO2 and silanized MagNPs
The magnetic nanoparticles were prepared by the co-
precipitation method, and their silanization was described
elsewhere [18].
∗
Corresponding author.
E-mail address: henetoma@iq.usp.br (H.E. Toma).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.03.021

C.G.C.M. Netto et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 136–143
137
2.2. Glyoxyl-agarose synthesis
The synthesis of glyoxyl-agarose was performed as described by
Manrich et al. [19].
2.3. General procedure of FDH immobilization on MagNP-APTS
The enzyme stock solution consisted of 3.13 mg/mL FDH in
Tris–HCl 0.1 M (pH 7.5) buffer. For the immobilization step, 300 ␮L
of this solution (0.94 mg, 5 U) was mixed with 500 ␮L of Tris–HCl
0.1 M buffer containing MagNP-APTS (1, 2, 4, 8 and 16 mg).
Then, glutaraldehyde (1.2 ␮mol, 2.4 ␮mol, 4.8 ␮mol, 9.6 ␮mol or
19.2 ␮mol) was added and allowed to react at 0 ◦C for a determined
period of time (0, 2, 5, 10, 15, 20 and 60 min). The immobilized
enzymes were confined by using an external miniature Nd2Fe14
B
magnet (1 cm3, 11 kOe from MagTek), and washed 3 times with
0.5 mL of Tris–HCl 0.1 M buffer solution. The best conditions were
obtained by using 16 mg of MagNP-APTS, 1.2 ␮mol of glutaralde-
hyde and 1 h of immobilization.
Fig. 1. Covalent binding of the enzyme (FDH) to magnetic particles via glutaralde-
hyde (structures not in scale).
2.4. General procedure of FDH immobilization on
MagNP@SiO2-APTS
20 ◦C. ꢀH =/ and ꢀS =/ measurements were performed at 20, 21, 22,
23 and 25 ◦C and pH 7.5.
As in the preceding case, MagNP@SiO2-APTS (1, 2, 4, 8, 13, 16
and 18 mg) was suspended into 0.5 mL of Tris–HCl 0.1 M buffer,
and 300 ␮L of the FDH stock solution (0.94 mg, 5 U) was added,
followed by 1.2% glutaraldehyde aqueous solution (0, 0.6 ␮mol,
1.2 ␮mol, 2.4 ␮mol, 4.8 ␮mol, 9.6 ␮mol or 19.2 ␮mol). The mix-
ture was allowed to react at 0 ◦C during 0, 2, 5, 10, 15 or 20 min.
After confining the immobilized enzymes by using an external
miniature magnet, the particles were washed 3 times with 0.5 mL
Tris–HCl 0.1 M of buffer solution. The best conditions were obtained
with 13 mg of MagNP@SiO2-APTS, 0.6 ␮mol of glutaraldehyde and
10 min of immobilization.
2.7. Physical measurements
UV–VIS spectrophotometry measurements were carried out on
a Hewlett Packard 8453-A diode-array spectrophotometer.
For atomic force microscopy (AFM) measurements 5 ␮L of
nanoparticle solution were deposited over mica (Ted Pella Inc.),
and allowed to dry in a clean laminar flow chamber. The AFM
images were obtained using a PicoSPM I microscope (Molec-
ular Imaging, MI) with PicoScan 2100 (MI) controller coupled
with MACMode (MI) unit for intermittent contact AFM and MAC
Mode SFM. Data acquisition were obtained using a PicoScan (M.I)
device with the scan rate between 0.5 and 1.0 Hz operating from
256 to 512 points per line. For the AFM and MACMode SFM
measurements, silicon tips with high aspect ratio from Nanosen-
sors and Agilent (Type II MACLevers, k ∼ 2.8 N/m; f ∼ 60 kHz) were
employed.
2.5. General procedure of FDH immobilization on
MagNP-APTS/Glyoxyl-Agarose
In this case, 8 mg of MagNP-APTS and 300 ␮L of the FDH stock
solution (0.94 mg, 5 U) were mixed with 700 ␮L of Tris–HCl buffer
solution (0.1 M) and 300 ␮L of glyoxyl-agarose (100 mg/mL) was
added. The immobilization was allowed to proceed for 10 min at
0 ◦C. Then, by using an external miniature magnet, the particles
were washed 3 times with 0.5 mL of buffer and finally dispersed
into 0.6 mL of the buffer solution.
Fluorescence measurements were recorded on a Photon Tech-
nology International Inc. model LS-100 spectrofluorometer.
3. Results and discussion
Magnetic nanoparticles obtained by the co-precipitation
method were silanized with aminopropyltriethoxysilane and
reacted with glutaraldehyde in order to form imino bounds
between the enzyme and nanoparticle. Both particles, MagNP-APTS
and MagNP@SiO2-APTS were submitted to similar immobiliza-
tion methodologies, differing only in the inner shell composition
and particle size, while the method using glyoxyl-agarose as a
“substitute” for glutaraldehyde, involved a multi-point covalent
immobilization.
2.6. Formate conversion to CO2
The magnetic-FDH was transferred into a cuvette containing
1 mL of Tris–HCl buffer solution (0.1 M). Then 10 ␮L of 0.4 M sodium
formate and 5 ␮L of NAD+ (0.9 mM) were added. The kinetics of
formation of NADH was monitored at 340 nm.
2.6.1. Temperature and pH dependence studies
pH dependence studies were performed at 37 ◦C. For the tem-
perature studies, the pH was kept at 7.5.
3.1. Optimization of the immobilization methodology
2.6.2. Recycling studies
The best FDH-magnetic particle system was achieved after sev-
eral immobilization optimizations, which resulted in a different
protocol for each support.
For the recycling studies, MagNP-APTS/FDH were used at pH
7.5 and 22 ◦C, MagNP@SiO2-APTS/FDH and MagNP-APTS/Glyoxyl-
Agarose/FDH were used at pH 7.5 and 27 ◦C, corresponding to their
best performance conditions.
3.1.1. Immobilization by glutaraldehyde
2.6.3. Thermodynamic studies
The immobilization of formate dehydrogenase on magnetic
nanoparticles by the glutaraldehyde method results in systems
similar to those shown in Fig. 1.
KM measurements were performed in 0.1 M Tris–HCl buffer, pH
7.5, employing 50 ␮L of NAD+ (0.9 mM) and 40 mM of formate, at

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Fig. 2. Relation between the amount of immobilized FDH (red line) and formate
conversion (black line) to the amount of MagNP-APTS in the immobilization process.
(For interpretation of the references to color in this figure legend, the reader is
referred to the web version of the article.)
Fig. 5. Relation between the amount of MagNP@SiO2-APTS added to the immobi-
lization process and the formate conversion.
For the MagNP@SiO2-APTS particles, by increasing their amount
in the immobilization process, keeping the same amount of FDH
(0.94 mg, 5 U), a gradual increase of conversion was observed up
to 13 mg of nanoparticles. After this point, the formate conversion
decreased, as shown in Fig. 5.
We observed two maximum peaks in relation to the immobiliza-
tion time, one at 2 min and the other at 10 (Fig. 6). For this reason,
the influence of the amount of glutaraldehyde was investigated for
the two periods of time. For the two minutes peak, the addition
of glutaraldehyde decreased the formate conversion, correspond-
ing to a possible adsorption of FDH on the particles surface, while
for 10 min of immobilization, 5 ␮mol of glutaraldehyde gives the
best conversion performance and suggests also the formation of
covalent bounds between enzyme and support (Fig. 7).
Fig. 3. Effect of the amount of glutaraldehyde on the formate conversion.
3.1.2. Immobilization by glyoxyl-agarose
The enzyme (FDH) was covalently attached to glyoxyl-agarose
and MagNP-APTS resulting in MagNP-APTS/Glyoxyl-Agarose/FDH,
as illustrated in Fig. 8.
Formate dehydrogenase immobilized on magnetic glyoxyl-
agarose support suffered a decrease in formate conversion with
the increase in immobilization time (Fig. 9). For this reason, the
best adopted protocol was the rapid mixture of the two compo-
nents. This short period of time was apparently enough for binding
the support and enzyme, producing no negative effect on the FDH
conformation.
The first variable to be analyzed was the amount of added
MagNP-APTS. Keeping the concentration of 0.2 M of glutaralde-
hyde and 5 min of immobilization time, we observed that 0.5 mg
of FDH was immobilized on 8 mg and 16 mg of particles; however,
the formate conversion increased by a factor of two when 16 mg of
MagNP-APTS were used (Fig. 2).
By increasing the amount of glutaraldehyde in the immobi-
lization process, the immobilized enzyme exhibited a decreased
activity, as shown in Fig. 3. Accordingly, we have chosen
1.5 × 10−5 mol of glutaraldehyde as the best value.
From the suspension obtained after the immobilization pro-
tocol, accurate volumes were employed for the reaction. It
was observed that the increase in concentration of MagNP-
Glyoxyl-Agarose/FDH in the reaction mixture leads to an increase
The increase in the immobilization time improved the yields of
formate conversion, reaching a maximum rate at 1 h (80% formate
conversion) as shown in Fig. 4. After this time, the enzyme activity
decreased drastically.
Fig. 4. Relation between the immobilization time and formate conversion.
Fig. 6. Immobilization time influence on the formate conversion.

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139
Fig. 10. MagNP-Glyoxyl-Agarose/FDH system: relation of enzymatic system
amount with formate conversion.
Fig. 7. Effect of the amount of glutaraldehyde on the formate conversion for 2 min
(red line) and 10 min of immobilization (black line). (For interpretation of the refer-
ences to color in this figure legend, the reader is referred to the web version of the
article.)
conglomerate. In contrast, for the MagNP@SiO2-APTS/FDH system
(Fig. 12) the observed poor phase contrast indicated that the mag-
netic nanoparticles are surrounded by the enzyme. In the case of
MagNP-APTS/Glyoxyl-Agarose/FDH, although the phase contrast
image shows some magnetic nanoparticles exposed at the sur-
face (Fig. 13B), presumably most of them should be covered by the
agarose and enzyme coating.
3.3. Temperature and pH dependence studies
These FDH-magnetic particles were used in catalytic studies at
different pH and temperatures. Free FDH seems to have a nearly
constant activity for formate conversion, around 40%, in the range
of pH from 5.8 to 8.8. In the case of MagNP@SiO2-APTS/FDH an opti-
mum pH around 7.5 is clearly defined, increasing the conversions
up to 90%. In contrast, for MagNP-APTS/Glyoxyl-Agarose/FDH the
conversion remains practically constant, around 20%, between pH
5.8 and 8.8. On the other hand, MagNP-APTS/FDH exhibits a con-
trasting catalytic profile, starting with negligible conversion rates
at low pHs, but increasing rapidly above pH 7.5 and reaching 90%
conversion at pH 9.8 (Fig. 14).
Fig. 8. Covalent binding of the enzyme (FDH) to magnetic particles using glyoxyl-
agarose.
in conversion, reaching 94% for 200 ␮L of MagNP-Glyoxyl-
Agarose/FDH (Fig. 10).
In the temperature dependence studies, although the best per-
formance for free FDH was 37 ◦C, corresponding to 35% of formate
conversion, this enzyme seemed quite robust to temperature vari-
ation (Fig. 15) since it didn’t loose much activity upon heating up to
47 ◦C (32% of formate conversion). When immobilized on MagNP-
APTS and MagNP@SiO2-APTS, formate dehydrogenase gave higher
conversions, but became more sensitive to temperature changes.
MagNP-APTS/FDH was more active at temperatures higher than
37 ◦C with an optimum temperature at 42 ◦C, while MagNP@SiO2-
APTS/FDH exhibited higher conversions in the 23–37 ◦C interval.
The immobilization through glyoxyl-agarose rendered FDH very
sensitive to the temperature, shifting the maximum conversion
temperature to 27 ◦C.
3.2. Morphology
The morphology of the films was analyzed by atomic force
microscopy in order to verify the homogeneity and size. After
drying over mica the AFM phase contrast image (Fig. 11B)
of MagNP-APTS/FDH revealed agglomerates of about 0.5–2 ␮m
comprising small inorganic clusters anchored on the enzyme
3.4. Recycle
When submitted to recycle, all the three systems exhibited
about 30% decay from the original activity after the first cycle, keep-
ing afterwards a constant activity, at least for the next five cycles.
The relative activity of MagNP@SiO2/FDH (green bars, Fig. 16) was
always significantly higher during the five initial cycles, while
MagNP-APTS/Glyoxyl-Agarose/FDH (red bars, Fig. 16) exhibited the
smallest activity. In both cases, a sharp decrease was observed
for the activity after the 6th cycle (Fig. 16). The MagNP-APTS/FDH
system (blue bars, Fig. 16) exhibited a more stable behavior, main-
taining its activity around 40% up to 10 successive cycles.
Fig. 9. MagNP-Glyoxyl-Agarose/FDH system: relation of immobilization time with
the formate conversion.

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Fig. 11. Atomic force microscopy images of MagNP-APTS/FDH (A) topography and (B) phase contrast.
Fig. 12. Atomic force microscopy images of MagNP@SiO2-APTS/FDH (A) topography and (B) phase contrast.
Fig. 13. Atomic force microscopy images of MagNP-APTS/Glyoxyl-Agarose/FDH (A) topography and (B) phase contrast.

C.G.C.M. Netto et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 136–143
141
Table 1
Difference of KM, ꢀH =/ and ꢀS =/ for free and immobilized FDH.
c
d
Activity (U)a
KM (mM)b
ꢀH =/
ꢀS =/
Free FDH
5
6
3
20
9
13
−78
−104
−115
−120
MagNP-APTS/FDH
MagNP@SiO2-APTS/FDH
MagNP-APTS/Glyoxyl
agarose/FDH
4
12
6
8.6
5.9
21
a
27 ◦C, pH 7.5.
b
20 ◦C, Tris–HCl buffer (0.1 M pH 7.5), 5 ␮L of NAD+ (0.9 mM) and 40 mM of for-
mate.
c
kJ mol−1
.
d
J mol−1 K−1
.
Fig. 14. Enzyme activity in relation to pH for free FDH (line D), MagNP-Glyoxyl-
Agarose/FDH (line C), MagNP-APTS/FDH (line A) and MagNP@SiO2-APTS/FDH (B).
Fig. 17. Enzyme inactivation at 60 ◦C and pH 7.5. Free FDH (ꢀ), MagNP-APTS/FDH
(ꢀ), MAgNP@SiO2-APTS/FDH (ꢀ) and MagNP-APTS/Glyoxyl-Agarose/FDH (ꢁ).
Fig. 15. Enzyme activity in relation to temperature for free FDH (blue line, D),
MagNP-Glyoxyl-Agarose/FDH (red line, C), MagNP-APTS/FDH (black line, A) and
MagNP@SiO2-APTS/FDH (green line, B). (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of the article.)
the smaller activation enthalpy was compensated by the increase
of KM, while in the case of MagNP-APTS/Glyoxyl-Agarose/FDH
all the factors involved are less favorable in relation to the free
enzyme.
3.5. Thermodynamic properties
Activation energy and entropy were obtained for the nanopar-
ticles/enzyme and free enzyme pursuing additional clues on how
the immobilization can influence the enzyme activity. As shown
in Table 1, the higher activity observed for MagNP-APTS/FDH is
accompanied by the decrease of the Michaelis–Menten constant,
KM by a factor of two. The observed decrease in the activation
energy would reflect a favorable influence of the catalytic site, how-
ever this is more than compensated by the decrease of activation
entropy, implying in a more organized structure. The best compro-
mise between the activation energy and entropy was observed for
MagNP-APTS/FDH system. For the MagNP@SiO2-APTS/FDH system,
Fig. 18. Scheme of FDH from Candida boidinii showing the tryptophans sites (red
circles). (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of the article.)
Fig. 16. Recycling, in sequence presentation, of MagNP-APTS/FDH (blue line)
MagNP-Glyoxyl-Agarose/FDH (red line) and MagNP@SiO2-APTS/FDH (green line),
respectively. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of the article.)
Adapted from [22].

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C.G.C.M. Netto et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 136–143
Fig. 19. Relation between the catalysis at different pH (blue line, ꢀ) and the magnitude of quenching from original tryptophan emission before and after addition of formate
(black line, ꢀ) and NAD+ (red line, ꢀ) for (A) free FDH, (B) MagNP-APTS/FDH, (C) MagNP@SiO2-APTS/FDH and (D) MagNP-APTS/Glyoxyl-Agarose/FDH. (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of the article.)
3.6. Enzyme stability
to probe the substrate binding, undergoing quenching and shifts
[20–23]. The difference in the emission intensity can be used for
comparing the enzyme catalysis at different pHs, before and after
the substrate and coenzyme addition.
In order to check for the enzyme stability, they were pre-
incubated for a period of time up to 1 h, at 60 ◦C and pH 7.5, and their
activity evaluated. The slope values for free FDH and MagNP@SiO2-
APTS were rather similar (−0.023 min−1), while MagNP-APTS/FDH
exhibited a smaller slope value (−0.020 min−1) indicating an
increase in FDH stability. MagNP-APTS/Glyoxyl-Agarose/FDH pre-
sented a slope value of −0.086 min−1, being completely inactive
after 20 min of incubation (Fig. 17).
Considering the FDH structure (PDB code, 2JCI), visualized with
the Discovery Studio 2.5 program (Accelerys, San Diego, CA), the
binding site possesses histidine, arginine and asparagine residues
capable of forming H-bond with the oxygen atoms from formate.
Histidine has already been proposed as a key residue directly
involved in formate binding [24]. In the case of NAD+ binding, while
isoleucine and valine provide a favorable hydrophobic environ-
ment, the adenine group can be sandwiched between an arginine
and a cysteine, which are considered essential residues for coen-
zyme binding [23]. Actually, these residues can also determine how
the catalysis will proceed at different pHs. In this sense, Blanchard
and Cleland [25] reported that the binding of NAD+ is prevented by
3.7. Structure–activity dependence
Tryptophan exhibits a characteristic fluorescence in the visi-
ble (from 300 to 350 nm) upon excitation at 280 nm. The emission
is particularly sensitive to local electric fields and has been used

C.G.C.M. Netto et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 136–143
143
the protonation of a group with pK = 6.4 and by deprotonation of
a group with pK = 8.4. Such groups might be related with histidine
and cysteine, respectively.
by MagNP-APTS, MagNP@SiO2-APTS and MagNP-APTS/Glyoxyl-
Agarose, resulted in a gain of enzymatic performance and stability.
The silica gel coated nanoparticles (MagNP@SiO2-APTS) provided
the highest conversion rates (90% of formate conversion); however,
in terms of recycling, magnetite without the silica shell (MagNP-
APTS) led to the most stable system, with formate conversions
around 40% for 10 successive cycles. MagNP-APTS/Glyoxyl-Agarose
was consistently less active than the other two systems, exhibit-
ing conversions of 30% and low temperature stability. By using
the enzyme tryptophan residues as internal fluorescence probes,
the structural–activity behavior was investigated in the presence
of the formate and NAD+ substrates, revealing a rather contrast-
ing behavior in the three cases. A close interaction of the magnetic
nanoparticles with the catalytic sites seems to be implicated in the
case of MagNP-APTS. In the case of the agarose modified nanoparti-
cle, the enzyme tryptophan residues seem to undergo non-specific
quenching, precluding any reliable correlation with the catalytic
sites.
Another important point, as shown in Fig. 18, is that formate
dehydrogenase from C. boidinii possesses 5 different tryptophans,
four of them quite near the binding sites for NAD+ and formate,
while the other one is located far away from the binding sites. These
tryptophans residues are indicated by red circles in Fig. 18.
As one can see in Fig. 19A, there is a good correlation between the
changes in the tryptophan emission and the free enzyme activity
after adding formate and NAD+, as a function of pH. The maximum
activity is observed at pH 7.5 suggesting the participation of non-
protonated histidine at this pH, in agreement with the hypothesis
of Blanchard and Cleland [25]. The observed correlation means that
the fluorescence changes from the tryptophan residues can effec-
tively be used to probe the formate and NAD+ binding sites in free
FDH.
In the case of the enzyme immobilized on MagNP-APTS
(Fig. 19B), the observed profiles are quite different from those of
the free enzyme (Fig. 19A). The catalytic conversion increases with
the pH (blue line, Fig. 19B), nearly paralleling the decay of the tryp-
tophan emission in the presence of formate (black line, Fig. 19B)
and NAD+, showing a negative fluctuation in this case, around pH
8.8, probably due to the deprotonation of the cysteine residue, as
observed by Blanchard and Cleland [25]. MagNP-APTS/FDH-NAD+
binary complex should have suffered an increase in its pKa (orig-
inally 8.3–8.5) [25] since it must be protonated for the formate
binding and the interaction of formate increases with increasing pH
in this immobilized form of formate dehydrogenase. The observed
tendency of increase with the pH (above pH 9) may reflect the
involvement of additional sites, such as the residual amine groups
from MagNP-APTS, since this effect is not observed for the free
enzyme.
Acknowledgements
The financial support from FAPESP and CNPq agencies is grate-
fully acknowledged.
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Immobilization of formate dehydrogenase from C. boi-
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