Catalytic properties of catechol 1,2-dioxygenase from Acinetobacter radioresistens S13 immobilized on nanosponges

Article abstract of DOI:10.1039/b903105g

Catechol 1,2-dioxygenases are iron containing enzymes able to convert catechol into cis,cis-muconate, a precursor of the industrially important compound adipic acid. Catechol 1,2-dioxygenase from Acinetobacter radioresistens S13 was immobilized on β-cyclodextrins cross-linked with carbonate groups (nanosponges) with a yield of 29 mg of enzyme per gram of support. This support was chosen for its low cost and its ability to offer different types of interactions with the enzyme. The activity profiles at different pH and temperatures showed a shift of the optimal pH from 8.5, for the free protein, to 9.5, for the immobilized protein and, similarly, a shift in optimal temperature from 30 °C to 50 °C. The Michaelis-Menten constant, K_M, increased from 2.0 ± 0.3 μM, for the free form, to 16.6 ± 4.8 μM for the immobilized enzyme, whereas the rate constant, k_cat, values were found to be 32 ± 2 s^-1 and 27 ± 3 s ^-1 for the free and immobilized forms respectively. The immobilization process also increased the thermostability of the enzyme with 60% residual activity after 90 min at 40 °C for the immobilized protein versus 20% for the free enzyme. A residual activity of 75% was found after 15 min at 60 °C for the immobilized enzyme while the free form showed a total loss of activity under the same conditions. The activity toward other substrates, such as 3- and 4-methylcatechol and 4-chlorocatechol, was retained by the immobilized enzyme. A small scale bioreactor was constructed and was able to convert catechol into cis,cis-muconic acid with high efficiency for 70 days. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b903105g

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Dalton
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An international journal of inorganic chemistry
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Number 33 | 7 September 2009 | Pages 6433–6668
ISSN 1477-9226
HOT ARTICLE
COMMUNICATION
Gilardi et al.
PERSPECTIVE
Xu, Cronin et al.
Catalytic properties of catechol
1,2-dioxygenase from Acinetobacter
radioresistens S13 immobilized on
nanosponges
Wang, Sun et al.
A functional hybrid polyoxometalate
Light-driven hydrogen production
catalysed by transition metal
complexes in homogeneous systems
framework based on a
‘trilacunary’heteropolyanion
[(P4W6O34)2Co2Na2(H2O)2]18−

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PAPER
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Catalytic properties of catechol 1,2-dioxygenase from Acinetobacter
radioresistens S13 immobilized on nanosponges
Giovanna Di Nardo,a Carlo Roggero,b Simona Campolongo,a Francesca Valetti,a Francesco Trottac and
Gianfranco Gilardi*a,d
Received 16th February 2009, Accepted 20th May 2009
First published as an Advance Article on the web 14th July 2009
DOI: 10.1039/b903105g
Catechol 1,2-dioxygenases are iron containing enzymes able to convert catechol into cis,cis-muconate,
a precursor of the industrially important compound adipic acid. Catechol 1,2-dioxygenase from
Acinetobacter radioresistens S13 was immobilized on b-cyclodextrins cross-linked with carbonate
groups (nanosponges) with a yield of 29 mg of enzyme per gram of support. This support was chosen
for its low cost and its ability to offer different types of interactions with the enzyme. The activity
profiles at different pH and temperatures showed a shift of the optimal pH from 8.5, for the free
protein, to 9.5, for the immobilized protein and, similarly, a shift in optimal temperature from 30 ◦C to
50 ◦C. The Michaelis–Menten constant, KM, increased from 2.0 0.3 mM, for the free form, to
16.6 4.8 mM for the immobilized enzyme, whereas the rate constant, kcat, values were found to be
32 2 s-1 and 27 3 s-1 for the free and immobilized forms respectively. The immobilization process
also increased the thermostability of the enzyme with 60% residual activity after 90 min at 40 ◦C for the
immobilized protein versus 20% for the free enzyme. A residual activity of 75% was found after 15 min
at 60 ◦C for the immobilized enzyme while the free form showed a total loss of activity under the same
conditions. The activity toward other substrates, such as 3- and 4-methylcatechol and 4-chlorocatechol,
was retained by the immobilized enzyme. A small scale bioreactor was constructed and was able to
convert catechol into cis,cis-muconic acid with high efficiency for 70 days.
of catechol resulting in the formation of cis,cis-muconic acid.5
Introduction
Catechol is a common intermediate in the degradation pathways
The exploitation of enzymes as biocatalysts for the “green”
of many monocyclic aromatic compounds, including benzene and
production of industrially important compounds is an interesting
toluene,6,7 and polycyclic aromatic molecules, such as pyrene8 and
challenge for biotechnology. One such process is the enzymatic
naphthalene.9,10
cleavage of catechol that leads to cis,cis-muconic acid which in
turn is hydrogenated to produce adipic acid, commonly used for
In order to construct a bioreactor able to produce muconic acid
with high efficiency and operational stability, the enzyme catechol
the benzene-free synthesis of nylon-6,6.1 The worldwide industrial
1,2-dioxygenase was selected as it does not require any external
production of adipic acid is estimated to be 2.3 million metric tons
per year and 80% of it is used for nylon-6,6 synthesis.2 The global
market for adipic acid has been predicted to reach 6.3 billion
cofactor for catalysis. In addition, in this work, the isoenzyme B of
catechol 1,2-dioxygenase from Acinetobacter radioresistens S1311,12
was immobilized on nanosponges formed by b-cyclodextrins
pounds by 2012 (report by Global Industry Analysts, Inc.) as
linked by carbonate groups to give a solid support.13 To our
it is also used as basic feedstock in the production of engineering
knowledge this is the first study where direct enzyme immobiliza-
plastics, polyurethane elastomers, pharmaceuticals and derivatives
tion has been performed on modified cyclodextrins with the aim
for clothes. Furthermore, adipic acid is also used for the synthesis
of creating a bioreactor. Recently, cyclodextrin derivatives have
of biodegradable polymers with applications in many processes
been used to construct supramolecular assemblies to increase the
including green polyurethane coatings3 and tissue engineering.4
number of adamantine-modified protein molecules at the surface
For these reasons our laboratory has turned its attention to
of gold electrodes,14–16 but these works were without the advantage
catechol 1,2-dioxygenases from Acinetobacter radioresistens, an
iron containing enzyme involved in the degradation of aromatic
of a sponge-like solid support suitable for the construction of a
bioreactor, as described in this paper.
compounds, including phenols. It catalyses the intradiol cleavage
One of the major current goals in the field of biocatalysis
is to develop low-cost systems using suitable enzymes that are
aDepartment of Human and Animal Biology, University of Torino, via
permanently immobilized on a solid support and are able to
perform different cycles of catalysis. Both catechol 1,2-dioxygenase
and nanosponges were selected here as they respond to these
requirements.13,17 In particular, nanosponges were chosen for
enzyme immobilization on the basis of their physico-chemical
properties. On the one hand, the hydrophobic cavities of the
b-cyclodextrins are known to accommodate solvent exposed
Accademia Albertina 12, 10123, Torino, Italy. E-mail: gianfranco.gilardi@
unito.it; Fax: +39 011 670 4643; Tel: +39 011 670 4593
bSea Marconi Technologies S.a.s., via Ungheria 20, 10093, Collegno, Torino,
Italy
cDepartment of I.F.M. Chemistry, University of Torino, via P. Giuria 7,
10125, Torino, Italy
dDivision of Biomolecular Sciences, Imperial College London, London, UK
SW7 2AY
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aromatic side chains of the protein.18 On the other hand, hydroxyl
groups are displayed on their exterior and carbonates are present
in the linkers between different cyclodextrin units, giving rise
to a complex network of polar groups able to interact via
hydrogen bonds and electrostatic interactions with polar side
chains of the protein. These protein–support interactions between
the nanosponges and the enzyme do not present the disadvantages
of a covalent immobilization, where the enzyme is forced into
a single conformation that may not provide the most efficient
catalysis. The sum of all the weak interactions taking place between
the enzyme and nanosponges is expected to lead to a tight binding
of the protein to the support allowing different cycles of catalysis
whilst immobilized. In addition, the support offers the advantage
of being produced via a cheap synthetic procedure, key to its
applicability in large scale industrial processes.
Determination of the maximal binding capacity of nanosponges
The maximal amount of enzyme retained by the support was
determined by incubating 1.7 mg of protein with 0.05 g of
nanosponges at 22 ◦C for 4 h and by calculating the residual
protein content in the washing solutions.
Effect of pH and temperature on the enzyme activity
The effect of pH on the activity of catechol 1,2-dioxygenase in
the free and immobilized forms was investigated in the pH range
5.5–10 at 30 ◦C.
The enzyme was incubated for 3 min in different buffers with the
desired pH and the reaction was then started with the addition of
200 mM of catechol. The buffer solutions used were: Mes (for pH
ranging from 5.5 to 7), HEPES (for pH ranging from 7 to 8.5) and
Ches (for pH ranging from 8.5 to 10). The buffer concentrations
were chosen appropriately to maintain constant ionic strengths.
Catechol 1,2-dioxygenase immobilized on nanosponges was
characterized in terms of stability at different pH and tempera-
tures, storage and kinetic properties. The stability of the bioreactor
was tested over time for different reaction cycles.
◦
The activity at different temperatures (from 10 to 60 C) was
monitored by incubating the enzyme for 3 min in a 50 mM HEPES
pH 8.0 buffer in the spectrophotometer cell, equipped with a
Peltier 89090A temperature control system (Agilent Technolo-
gies). The reaction was then started with the addition of 200 mM
of catechol and the activity measured.
Experimental
Materials
The activation energies (Ea) were calculated by using the
Arrhenius equation:
Catechol 1,2-dioxygenase was expressed and purified as previously
described19 whereas cyclodextrin-based nanosponges were synthe-
sized as reported by Cavalli et al.17
ln k = -Ea/RT + ln A
where k is the rate constant, T is the temperature (K), R the gas
constant and A the pre-exponential factor.
Chemicals including catechol and substituted catechols were
purchased from Sigma-Aldrich.
Fittings of the experimental data were performed by using the
Sigma Plot 8.0 software.21
Determination of the protein concentration and activity assay
Thermostability and storage stability
Protein concentration was estimated at 280 nm by using an
extinction coefficient, e280, of 53 560 M-1 cm-1.12
The thermal stability was investigated by incubating the enzyme
in a 50 mM HEPES buffer at 2 different temperatures (40 ◦C and
60 ◦C). Aliquots were removed at regular intervals and assayed for
their residual activity.
Storage stability was investigated by monitoring the residual
activity of both free and immobilized enzymes after storing them
at 4 ◦C and 25 ◦C.
The concentration of active protein was determined from its
specific activity and was evaluated for each batch of protein used.
The enzyme activity was assayed by recording the absorbance
at 260 nm due to the formation of cis,cis-muconic acid.11 The
slope of the initial linear increase in absorbance at 260 nm was
considered. Assays were carried out at 30 ◦C in a 1 cm path-length
cell. The reaction mixture contained the enzyme in nanomolar
quantity and 200 mM catechol in a 50 mM HEPES pH 8.0 buffer.
The substrate, catechol, was freshly prepared in a 50 mM HEPES
pH 8.0 buffer solution. The product was quantified by using an
extinction coefficient at 260 nm (e260) of 17 600 M-1 cm-1.
Determination of the catalytic parameters
The catalytic parameters (Michaelis–Menten constant, KM, and
rate constant, kcat) of the free and immobilized enzymes were
calculated by measuring the initial linear rates of the reaction
after the addition of different concentrati◦ons of the substrate,
catechol, ranging from 1 to 100 mM at 30 C. Five independent
measurements were carried out for each substrate concentration.
The experimental data were fitted to the Michaelis–Menten curve
by using the Sigma Plot 8.0 software.21
Immobilization of catechol dioxygenase
The dried nanosponges were hydrated and washed five times with
100% ethanol and ten times with 50 mM HEPES pH 8.0. The
resuspended nanosponges (1 g) were gently mixed with enzyme
of a known concentration (0.2 mg) in a final volume of 1 mL at
Substrate specificity
◦
22 C. The supernatant was then removed and the nanosponges
washed with 50 mM HEPES pH 8.0 until no catechol 1,2-
dioxygenase activity was detected in the washing solutions.
The yield of immobilization was determined from the difference
between the initial quantity of protein loaded onto the support and
the quantity found in the washing solutions.
The ability of immobilized catechol 1,2-dioxygenase to recognise
3- and 4-methylcatechol and 4-chlorocatechol was measured
spectrophotometrically by monitoring the absorbance at 260 nm
due to the formation of the reaction product. The relative activity
was calculated, assuming the activity toward catechol to be 100%.
6508 | Dalton Trans., 2009, 6507–6512
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Table 1 Enzymatic parameters calculated for free and immobilized
catechol 1,2-dioxygenase
The substrate concentration was 200 mM, which was known to be
saturating and not inhibiting for the enzyme.
Free catechol
1,2-dioxygenase
Immobilised catechol
1,2-dioxygenase
Reusability
A solution containing 1 g of nanosponges and 0.3 mg of catechol
1,2-dioxygenase was used as the stationary phase to pack a column
(5 cm height, 1 cm diameter, GE Healthcare). The column was
stored at 4 ◦C. At regular intervals (1 day) 1 mM catechol (1 ml)
was loaded onto the column and, after 1 h, the column was
washed with 50 mM HEPES pH 8.0. The absorption spectra of the
washing solutions were recorded and the product, cis,cis-muconic
acid, was quantified by using an extinction coefficient (e260) of
17 600 M-1 cm-1.11
K
M/mM
2.0 0.3
16.6 4.8
k
cat/s-1
32
8.5
30
2
27
9.5
50
3
pH for maximal activity
Temperature for maximal
activity/◦C
Ea/kJ mol-1
45
5
Ea1 = 49
Ea2 = 19
2
3
Results
Immobilisation of catechol 1,2-dioxygenase on nanosponges
The purified catechol 1,2-dioxygenase was immobilized by ad-
sorption on nanosponges and the yield of immobilization was
evaluated by incubating, for different lengths of times, 0.3 mg of
enzyme with 1 g of solid support at 4 ◦C and 22 ◦C. As shown in
Fig. 1, 96% of the enzyme was immobilized onto the support after
4 h at 22 ◦C; a yield of 50% was reached after 10 min of incubation,
indicating a high affinity of the enzyme for the support.
Fig. 2 pH profile of catechol 1,2-dioxygenase activity in the free (white
squares) and immobilized (black circles) forms. The enzyme was incubated
for 3 min in different buffers with the desired pH and the reaction was then
started with the addition of 200 mM of catechol. The activity was measured
as the absorbance at 260 nm, due to the formation of cis,cis-muconic acid.
The initial linear increase was calculated over the first 2 min. The data
points represent the average of 5 independent measurements.
(Table 1). Furthermore, the immobilized enzyme showed 40%
activity at pH 6.5 whereas the free form was inactive.
The effect of temperature on enzyme activity was investigated in
the range 10–60 ◦C.◦The free enzyme exhibited optimum activity
◦
in the range 30–40 C with a complete loss of activity at 60 C,
while the immobilized enzyme showed the highest activity at 50 ◦C
Fig. 1 Time-course of immobilization of catechol 1,2-dioxygenase on
nanosponges at 22 ◦C. The quantity of protein immobilized is expressed
in %, where a yield of 100% represents the complete immobilization of the
enzyme initially loaded (0.2 mg) onto the support.
and retained 70% of its activity at 60 ◦C (Fig. 3A).
Arrhenius plots were then constructed and a linear relationship
was found up to 30 ◦C (303 K) and 60 ◦C (333 K) for the free and
immobilized enzymes respectively (Fig. 3B). As the temperature
was further increased, a drop in enzyme activity was observed,
indicating protein denaturation. However, a biphasic behaviour
was observed for the immobilized protein, with a discontinuity at
25 ◦C.
The experimental data were then fitted with linear regressions to
calculate the activation energies (Ea) from the Arrhenius equation
(Fig. 3B) and the results are shown in Table 1. The activation
energies were similar for the free and immobilized catechol
dioxygenase below 25 ◦C (45 5 kJ mol-1 and 49 2 kJ mol-1
respectively), whereas a lower Ea (19 3 kJ mol-1) was found for
the immobilized enzyme above 25 ◦C.
The maximal binding capacity of nanosponges was then evalu-
ated by mixing 1.69 mg of protein with 0.05 g of nanosponges for
4 h at 22 ◦C in a 50 mM HEPES pH 8.0 buffer. The nanosponges
were extensively washed with the buffer solution and the unbound
protein was quantified. The presence of catechol 1,2-dioxygenase
activity in the first washing solutions demonstrated that the protein
loaded was in excess with respect to the binding capacity of the
support, and resulted in 28.9 mg of protein per 1 g nanosponges.
Effect of pH and temperature on enzyme activity
The activity of free and immobilized catechol 1,2-dioxygenase was
measured at different pH values (from 5.5 to 10). As shown in
Fig. 2, the activity profiles for the free and immobilized proteins
were different and the pH value of optimum activity shifted from
8.5, for the free enzyme, to 9.5, for the immobilized enzyme
Thermostability and storage stability
The stability of the free and immobilized enzymes over time was
measured at 40 ◦C and 60 ◦C. Fig. 4A shows a complete loss
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Fig. 3 (A) Temperature profile of catechol 1,2-dioxygenase activity in the
free (white squares) and immobilized (black circles) forms. (B) Arrhenius
plot for the free (white squares) and immobilized (black circles) catechol
1,2-dioxygenase. The enzyme was incubated at the desired temperature for
3 min in a 50 mM HEPES pH 8.0 buffer. The activity was measured as the
absorbance at 260 nm, due to the formation of cis,cis-muconic acid. The
initial linear increase was calculated over the first 2 min. The data points
represent the average of 5 independent measurements.
Fig. 4 Thermostability at (A) 40 ◦C and (B) 60 ◦C of free (white squares)
and immobilized (black circles) catechol 1,2-dioxygenase. The enzyme
was incubated at the desired temperature in a 50 mM HEPES pH 8.0
buffer and aliquots were removed at regular intervals and assayed for their
residual activity. The data points represent the average of 5 independent
measurements.
◦
of activity after 90 min at 40 C in the case of the free enzyme,
whereas after the same time, the immobilized form retained 50%
of the maximal activity and became totally inactivated only after
200 min. Furthermore, the activity profile of the immobilized
protein shows a biphasic behaviour that might indicate the
presence of two enzymatic conformations, stabilized differently
by the immobilization process.
Determination of the catalytic parameters and substrate specificity
In order to calculate the apparent values of the Michaelis–Menten
parameters (KM and kcat), the activities of the free and immobilized
enzymes were measured at different substrate concentrations.
The reaction was followed at 260 nm and the initial linear rate
considered. A typical hyperbolic trend was observed for both free
and immobilized proteins (Fig. 5). Fittings of the experimental
data to the Michaelis–Menten curve resulted in a KM of 2.0 0.3
and 16.6 4.8 mM and a kcat of 32 2 and 27 3 s-1 for the free
and immobilized enzyme respectively. As reported in Table 1, an
increase in KM (from 2.0 0.3 to 16.6 4.8 mM) was found for the
immobilized protein whereas the kcat values were very similar for
the free and immobilized forms.
Fig. 4B shows the thermal stability profile at 60 ◦C. In this case
a complete loss of activity was observed after 15 min in the case
of the free enzyme whereas the immobilized form retained more
than 70% of the activity after the same time.
The storage stability was measured at 4 ◦C and 25 ◦C. After
10 days at 4 ◦C, the immobilized enzyme was demonstrated to
retain 50% of the initial activity, whereas the activity of the free
form was lower than 10% of the initial.
Similar results were found by storing the protein at 25 ◦C. The
free catechol dioxygenase retained 10% of the starting activity after
4 days, whereas for the immobilized form the residual activity was
50%. Complete inactivation was observed after 5 days for the free
enzyme and after 11 days for the immobilized protein. In these
measurements, a biphasic behaviour was again observed for the
immobilized enzyme, but not for the free form.
The relative activity on substituted 3- and 4-methylcatechol
and 4-chlorocatechol by the immobilized enzyme was found
to be similar for the first two compounds and
3 fold
higher for 4-chlorocatechol when compared to the free form
(Table 2).
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Table 2 Substrate specificity of catechol 1,2-dioxygenase in free and
immobilized forms
Relative activity (%)
(free catechol
1,2-dioxygenase)
Relative activity (%)
(immobilized catechol
1,2-dioxygenase)
Substrate
Catechol
100
100
3-Methylcatechol
4-Methylcatechol
4-Chlorocathecol
4.1 1.6
37.5 6.3
5.2 2.7
9.9 5.2
44.0 9.0
15.6 3.9
Fig.
6
Reusability of catechol 1,2-dioxygenase immobilized on
nanosponges. The percentage of product formed after 1 h at room
temperature from a 1 mM solution of the substrate, catechol, is shown
as a function of time (days). One reaction cycle a day was performed.
the polar groups (hydroxyls and carbonates) of the support can
interact with polar residues of the protein and form hydrogen
bonds. Although the structure of catechol 1,2-dioxygenase from
Acinetobacter radioresistens is not available, it is most probable
that all of these interactions can occur with aromatic and polar
residues present in the sequence of the protein.19 Sequence
alignment of the enzyme used in this work with that of catechol
1,2-dioxygenase from Acinetobacter sp. ADP1 revealed 71% of
sequence homology between the two proteins. The analysis of
the known crystal structure of catechol 1,2-dioxygenase from
Acinetobacter sp. ADP120 shows the presence of tryptophans,
tyrosines and phenylalanines spread on the protein surface. Most
of them are conserved in the sequence of our protein and could
potentially interact with nanosponges.
It is well known that immobilized enzymes can show a higher
stability than their free counterparts. For this reason, we investi-
gated the activity of the enzyme over a wide range of pH values
and temperatures and we found a shift of the maximal activity
to a higher pH value for the immobilized enzyme in comparison
to the free counterpart. An opposite behaviour was previously
observed for catechol 1,2-dioxygenase from Nocardia sp. NCIB
10503 immobilized on cyanogen bromide-activated agarose, where
the optimal pH was found in the 8–10 range for the free enzyme
and at 7.5 for the immobilized form.22
Also, in the case of the temperature profile (Fig. 3), a shift in
the optimal temperature from 30 ◦C for the free to 50 ◦C for
the immobilized enzyme was observed. This kind of behaviour
has already been reported for both intradiol catechol dioxygenase
entrapped on another support (calcium alginate hydrogels)23 and
extradiol catechol dioxygenase covalently immobilized by multi-
point attachment on highly activated glioxyl agarose beads.24
A shift in the optimal temperature can be explained by the
increase in conformational stability given to the protein by the
support. For the free catechol 1,2-dioxygenase used in this work,
it was previously demonstrated that structural changes of the
enzyme and loss of catalytic iron from the active site are linearly
proportional.12 As such, the interaction with the support could
decrease the mobility of the protein, allowing a tighter binding of
the catalytic iron to the active site of the enzyme.
Fig. 5 Michaelis–Menten plot for the immobilized catechol 1,2-dioxyge-
nase. The activity was monitored via the absorption at 260 nm. The initial
linear rate was considered at the different substrate concentrations. The
data points represent the average of 5 independent measurements.
Reusability
In order to assess the applicability of the enzyme–support system
in a bioreactor, 1 g of nanosponges carrying 0.3 mg of enzyme was
used to pack a column and the ability of the immobilized enzyme to
support different cycles of reaction was tested as a function of time.
For each cycle a solution of 1 mM catechol was injected through
the column and after 1 h the column was washed with buffer
and the amount of cis,cis-muconic acid was quantified by UV
spectroscopy at 260 nm. As shown in Fig. 6, the system converted
83% of the catechol to muconic acid in 1 h after 10 cycles and
40% after 60 cycles. The presence of catechol dioxygenase activity
was assayed from the solution from the column. No activity was
found.
Discussion
The enzyme, catechol 1,2-dioxygenase, was successfully immo-
bilized by adsorption on nanosponges, a support that offers
the opportunity of use in the field of protein immobilization
for the development of biocatalysts suitable for industrial scale
production of muconic acid. The binding of the protein is ex-
plained by different types of interactions. In fact, literature NMR
data about the interaction between proteins and cyclodextrins in
solution18 showed that the hydrophobic cavity of cyclodextrins can
accommodate the aromatic side chains of solvent exposed residues,
such as tryptophans, tyrosines and phenylalanines. Furthermore,
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The experimental data obtained from the temperature profiles
were used to construct Arrhenius plots that revealed a biphasic
behavior for the immobilized protein, with a decrease in the
activation energy starting from 25 ◦C. Biphasic Arrhenius plots
can arise due to a number of effects, including protein denaturation
at high temperatures, mass transfer limitations, presence of two
different conformations of the enzyme stabilized in different
temperature ranges and a change in the rate determining step
of the reaction across the two temperature ranges.25,26 Enzyme
denaturation at high temperatures was tested by measuring the
residual activity against time at 40 ◦C and 60 ◦C; the immobilized
enzyme was more stable than the free counterpart, with a complete
loss of activity after 90 min at 60 ◦C. These results clearly indicated
that the biphasic Arrhenius plot obtained for the immobilized
enzyme could not be simply explained by protein denaturation at
high temperatures.
range of pH was improved and the small bioreactor engineered
was found to be stable over a period of 70 days without enzyme
detachment. The bioreactor presents all the features required to
be upgraded to a continuous-flow system that could maximize the
production of muconic acid. Such an engineered system would be
a useful tool to obtain an expensive compound such as muconic
acid, from the much cheaper catechol, for the production of the
industrially important adipic acid.
Acknowledgements
We thank R. Caglio for technical support in the enzyme purifi-
cation. Dr Giovanna Di Nardo is grateful for the award of a
Lagrange fellowship (CRT and ISI Foundations) and Sea Marconi
Technologies s.a.s. for financial support.
References
As stated before, mass transfer limitations can also give rise
to biphasic Arrhenius plots and a decrease of the activation
energy after immobilization has already been observed for
other enzymes27 and correlated to intraparticle diffusion of the
substrate,28 consistent with the presence of a pore diffusion effect.29
In the case of immobilized catechol 1,2-dioxygenase, the activation
energy calculated below 25 ◦C was comparable to that of the
free enzyme (Table 1) whereas it was significantly decreased above
25 ◦C. A possible effect of the support used for the immobilization
was excluded because it is known that nanosponges are chemically
1 W. Niu, K. Draths and J. Frost, Biotechnol. Prog., 2002, 18, 201–211.
2 A. Shimizu, K. Tanaka and M. Fujimori, Chemosphere glob chang sci,
2000, 2, 425–434.
3 H. R. Moeini, J. Appl. Polym. Sci., 2007, 106, 1853–1859.
4 K. Hemmrich, J. Salber, M. Meersch, U. Wiesemann, T. Gries, N. Pallua
and D. Klee, J. Mater. Sci.: Mater. Med., 2008, 19, 257–267.
5 C. Nakai, K. Horiike, S. Kuramitsu, H. Kagamiyama and M. Nozaki,
J. Biol. Chem., 1990, 265, 660.
6 P. Williams and M. Worsey, J. Bacteriol., 1976, 125, 818.
7 C. Winstanley, S. Taylor and P. Williams, Mol. Microbiol., 1987, 1,
219–227.
8 S. Kim, O. Kweon, R. Jones, J. Freeman, R. Edmondson and
C. Cerniglia, J. Bacteriol., 2007, 189, 464–472.
◦
and physically stable up to 120 C.30 In order to test if mass
9 M. Zeinali, M. Vossoughi and S. Ardestani, J. Appl. Microbiol., 2008,
105, 398–406.
transfer limitations could affect our system, we compared the
catalytic parameters of the free and immobilized enzyme. Even
if an increase in the KM value for the immobilized enzyme was
observed, the kcat was not affected, excluding the influence of pore
diffusion effects31 and of a change in the rate determining step of
the reaction across the two temperature ranges, both of which are
known to be possible causes of a biphasic Arrhenius plot.
These data, together with the biphasic behaviour found in
the thermostability profiles (Fig. 4A), strongly suggest that two
different conformations of the enzyme are present on the support
and that a conformational transition could take place at 25 ◦C. The
support would stabilize two different forms of the enzyme, depend-
ing on the conditions used, and give conformational constraints
that would explain also the change in KM observed and the increase
in activity of the immobilized form toward 4-chlorocatechol.
The enzyme–support system obtained from the immobilization
of catechol dioxygenase on nanosponges was also tested for its
reusability and showed the ability to convert 50% of the substrate,
catechol, after 50 cycles without enzyme detachment from the
support. These results demonstrate not only the reusability of the
system for different cycles, but also that the enzyme is tightly
bound to the support despite the non-covalent immobilization
method used.
10 M. Zeinali, M. Vossoughi and S. Ardestani, Chemosphere, 2008, 72,
905–909.
11 F. Briganti, E. Pessione, C. Giunta, R. Mazzoli and A. Scozzafava,
J. Protein Chem., 2000, 19, 709–716.
12 G. Di Nardo, S. Tilli, E. Pessione, M. Cavaletto, C. Giunta and
F. Briganti, Arch. Biochem. Biophys., 2004, 431, 79–87.
13 F. Trotta and W. Tumiatti, It. Pat. WO 03/085002, 2003.
14 C. Camacho, J. Matias, R. Cao, M. Matos, B. Chico, J. Hernandez,
M. Longo, M. Sanroman and R. Villalonga, Langmuir, 2008, 24, 7654–
7657.
15 A. Fragoso, J. Caballero, E. Almirall, R. Villalonga and R. Cao,
Langmuir, 2002, 18, 5051–5054.
16 R. Villalonga, A. Fujii, H. Shinohara, Y. Asano, R. Cao, S. Tachibana
and P. Ortiz, Biotechnol. Lett., 2007, 29, 447–452.
17 R. Cavalli, F. Trotta and W. Tumiatti, J. Inclusion Phenom. Macrocyclic
Chem., 2006, 56, 209–213.
18 F. Aachmann, D. Otzen, K. Larsen and R. Wimmer, Protein Eng., Des.
Sel., 2003, 16, 905–912.
19 P. Caposio, E. Pessione, G. Giuffrida, A. Conti, S. Landolfo, C. Giunta
and G. Gribaudo, Res. Microbiol., 2002, 153, 69–74.
20 M. W. Vetting and D. H. Ohlendorf, Structure, 2000, 8, 429–440.
21 J. Gurevitch, Q. Rev. Biol., 1991, 66, 115–116.
22 M. R. Smith, C. Ratledge and S. Crook, Enzyme Microb. Technol.,
1990, 12, 945–949.
23 E. Kalogeris, Y. Sanakis, D. Mamma, P. Christakopoulos, D. Kekos
and H. Stamatis, Enzyme Microb. Technol., 2006, 39, 1113–1121.
24 R. Fernandez-Lafuente, J. Guisan, S. Ali and D. Cowan, Enzyme
Microb. Technol., 2000, 26, 568–573.
25 J. Londesborough, Eur. J. Biochem., 1980, 105, 211–215.
26 S. Mehrotra and H. Balaram, Biochim. Biophys. Acta, 2008, 1784, 2019.
27 A. David, N. Wang, V. Yang and A. Yang, J. Biotechnol., 2006, 125,
395–407.
28 K. Miyamoto, T. Fujii, N. Tamaoki, M. Okazaki and M. Y. Miura,
J. Ferment. Technol., 1973, 51, 566.
29 T. Bahar and A. Tuncel, J. Appl. Polym. Sci., 2002, 83, 1268–1279.
30 F. Trotta, unpublished work.
31 A. S. Bommarius and B. R. Riebel, in Biocatalysis. Fundamental and
Applications, ed. A. S. Bommarius, Wiley-VCH, Weinheim, pp. 113–
118.
Conclusions
In conclusion, this work shows the feasibility of an enzyme
based bioreactor for the production of muconic acid. The enzyme
catechol 1,2-dioxygenase, which does not require an external
cofactor for catalysis, was successfully immobilized by adsorption
on a support formed by chemically modified cyclodextrins. The
stability of the enzyme at high temperatures and over a wide
6512 | Dalton Trans., 2009, 6507–6512
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