Oxidation of phenyl compounds using strongly stable immobilized-stabilized laccase from Trametes versicolor

Article abstract of DOI:10.1016/j.procbio.2013.05.021

The hydrolysis of phenolic compounds using an immobilized and highly active and stable derivative of laccase from Trametes versicolor is presented. The enzyme was immobilized on aldehyde supports. For this, the enzyme was enriched in amino groups by chemical modification of its carboxyl groups. The aminated enzyme was immobilized with a high recovered activity (over 60%). Aldehyde derivatives were more stable than soluble or aminated-soluble enzyme and the reference derivatives after incubation in different inactivating conditions (high temperatures, different pH values or presence of organic cosolvents). The most stable derivative was obtained immobilizing the chemically aminated enzyme at pH 10 on aldehyde supports with a stabilization factor approximately 280 fold after incubation at pH 7 and 55 C. In addition, it was possible to prepare immobilized derivatives with a maximal enzyme loading of 60 mg g^-1 of support. This derivative could be reused for 10 reaction cycles with negligible lost of activity.

Full text of DOI:10.1016/j.procbio.2013.05.021

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Contents lists available at SciVerse ScienceDirect
Process Biochemistry
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Oxidation of phenyl compounds using strongly stable
immobilized-stabilized laccase from Trametes versicolor
Veria Addorisioa,b, Filomena Sanninob, Cesar Mateoa,∗, Jose M. Guisana,∗
a Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain
b Dipartimento di Agraria, Università di Napoli “Federico II”, Via Università 100, 80055 Portici, Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The hydrolysis of phenolic compounds using an immobilized and highly active and stable derivative of
laccase from Trametes versicolor is presented. The enzyme was immobilized on aldehyde supports. For
this, the enzyme was enriched in amino groups by chemical modification of its carboxyl groups. The
aminated enzyme was immobilized with a high recovered activity (over 60%). Aldehyde derivatives were
more stable than soluble or aminated-soluble enzyme and the reference derivatives after incubation in
different inactivating conditions (high temperatures, different pH values or presence of organic cosol-
vents). The most stable derivative was obtained immobilizing the chemically aminated enzyme at pH
10 on aldehyde supports with a stabilization factor approximately 280 fold after incubation at pH 7 and
55 ◦C. In addition, it was possible to prepare immobilized derivatives with a maximal enzyme loading of
60 mg g−1 of support. This derivative could be reused for 10 reaction cycles with negligible lost of activity.
Received 14 January 2013
Received in revised form 23 April 2013
Accepted 30 May 2013
Keywords:
Enzyme immobilization
Trametes versicolor Laccase
Enzyme stabilization
Phenolic compound oxidation
© 2013 Elsevier Ltd. All rights reserved.
1. Introduction
or dye compounds into other more easily degradable by the
active sludge in the treatment plants [8]. Laccases (EC 1.10.3.2,
Several types of industrial and agricultural wastes contain
phenol compounds. For example, chlorophenols, largely used as
wide-spectrum biocides and nitrophenols, widely employed in the
chemical industry, accumulate in soils, sediments, surface waters
and animals because of their continuous usage and recalcitrant
nature [1]. Moreover, a lot of phenol compounds are recognized
as endocrine disruptors since they possess estrogenic or antie-
strogenic activities so interfering with the endocrine system [2].
Generally, these recalcitrant compounds produced in industries as
paper, oil or different ink industries cannot be degraded by the
active sludge in the treatment plants. Because of that the general
trend is treating these compounds with different enzymes in order
to transform them into degradable compounds or other non-toxic
ones [3]. The use of enzymes in these processes has a lot of advan-
tages because of their high activity in environmental conditions
such as room temperature, neutral pH or atmospheric pressure,
their high selectivity and specificity. One of the enzymes are being
used extensively is laccase.
p-diphenol: dioxygen oxidoreductase) catalyze the oxidation of a
variety of organic compounds including methoxyphenols, phenol,
o- and p-diphenols, aminophenols, polyphenols, polyamines, and
lignin-related molecules to o-, m-, p-quinones or radical species [9]
not requiring hydrogen peroxide as co-substrate or any cofactors
for its catalytic reaction. This makes this enzyme of great impor-
tance in different processes such as lignin modification [10], paper
strengthening [11,12], juice manufacture [13], hair coloring [14] or
in other different environmental processes [15,16]. Laccase from
Trametes versicolor is one of the most widely used in various pro-
cesses such as described in the literature [17–20]. It is a monomeric
enzyme of approximately 70 KDa of molecular weight [21].
However, the use of enzymes is not always easy on an industrial
scale because they have a number of limitations due to their bio-
logical origin. Thus enzymes are soluble and they cannot easily be
used for many reaction cycles; they are quite unstable under con-
ditions of high temperatures, extreme pH values or use of organic
co-solvents or toxic products. The availability of a strongly active
and stable biocatalyst is key for the application of these processes
at industrial scale. One way to avoid these problems consists in the
immobilization of enzymes. Immobilization promotes the reusabil-
ity of the biocatalyst during different reaction cycles. This enzyme
was previously immobilized using different methodologies. Some
of them immobilized this enzyme through different systems such as
entrapment in different polymers as alginate [22,23], dendrimers
Laccases are produced from various sources [4–7]. This enzyme
is able to oxidize different aromatic compounds such as phenol
∗
Corresponding authors. Tel.: +34 915854809; fax: +34 915854760.
E-mail addresses: ce.mateo@icp.csic.es (C. Mateo), jmguisan@icp.csic.es
(J.M. Guisan).
1359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2013.05.021
Please cite this article in press as: Addorisio V, et al. Oxidation of phenyl compounds using strongly stable immobilized-stabilized laccase from
Trametes versicolor. Process Biochem (2013), http://dx.doi.org/10.1016/j.procbio.2013.05.021

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using poly(amido amine) (PAMAM) [24] or gelatin [25], prepara-
tion of different systems as liposomes [26], microcapsules [27]
or cross-linked enzyme aggregates (CLEAs) [28]. However, the
most used system consisted in using different supports to immo-
bilize the enzyme. There are a great variety of supports used for
the immobilization as Amberlite IR-120H beads [29], membranes
[30], oxidized carbon nanotubes and graphene oxides [31,32], sulfo
polyester resins [33], exchange resins [34], magnetic nanoparticles
[35–37]. Among all these methods, the recovered activity is differ-
ent depending on the immobilization protocol, but the stabilization
generally is moderated and lowly studied. Best reported examples
give stabilization around 10–15 fold better than soluble enzyme
[19,38].
Taking into account the low stability of the soluble enzyme and
necessity of obtaining more stable catalysts, in the present paper
the immobilization of a commercial laccase from T. versicolor using
aldehyde supports is proposed. The optimized catalyst in terms of
activity and stability will be used in the oxidation of some phenolic
compounds.
enzyme activity in order to avoid diffusion problems that could alter the apparent
activity and/or enzyme stability.
The immobilization on cyanogen bromide Sepharose was performed as in the
case of aldehyde supports. In this case immobilization was carried out at 4 ◦C and pH
7 during short times. In the indicated cases this derivative was aminated by adding
to 1 g of laccase derivative 10 mL of EDA (1 M) and of EDAC (10 mM) at pH 4.75
during 90 min. Finally, this preparation was washed with water.
2.2.5. Stability of biocatalysts
Various preparations were incubated in different indicated conditions. Periodi-
cally, residual activity was determined as described above.
Inactivation was modeled based on the deactivation theory proposed by Henley
and Sadana [42]. Inactivation parameters were determined from the best-fit model
of the experimental data which was the one based on two-stage series inactiva-
tion mechanism with no residual activity. According to it, biocatalyst inactivation
proceeds through two sequential steps of progressively less active enzyme species
until a final completely inactive species is obtained, as represented in the following
scheme:
k
k
2
1
E−→ E1−→ E2
where k1 and k2 are first-order transition rates constants, E, E1 and E2 are the cor-
responding enzyme species. The mathematical model representing this mechanism
is
ꢀ
ꢀ
ꢁꢁ
ꢀ ꢀ
ꢁꢁ
2. Materials and methods
k1
k1
·t)
·t)
exp(−k
2
exp(−k
−
˛
(1)
1
a = 1 + ˛
k2 − k1
k2 − k1
2.1. Materials
where ˛ represents the residual activity at time t and ˛ is the ratio of specific activity
of enzyme species E1 with respect of that of the native enzyme species E.
Considering only one step mechanism of enzyme inactivation (k2 = 0), and resid-
ual activity (˛ =/ 0), the model is first order inactivation with residual activity,
represented by
Laccase from T. versicolor (2.2 IU mg−1), 2,2ꢀ-azino-bis(3-ethylbenzathiazoline-
6-sulfonic acid) (ABTS), ethylene diamine (EDA), sodium borohydride, sodium meta-
periodate, ethanolamine, glycidol (2,3-epoxypropanol), 2,6-dichloro-indophenol,
pyrogallol and catechol and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDAC) were supplied by Sigma Chem. Co. (St. Louis, MO, USA). Sepharose 10BCL
supports and cyanogen bromide (CNBr) activated Sepharose were supplied by GE
Healthcare (Little Chalfont, Buckinghamshire, UK). Cellulose dialysis tubes (cut-off
12–14 KDa) were from Spectrum Labs (Breda, The Netherlands). Other reagents were
always of analytical degree.
a = (1 − ˛)exp(−k
+ ˛
(2)
·t)
1
Inactivation parameters were determined from the best-fit model of the experi-
mental data. Half-life (time at which the residual enzyme activity is half of its initial
value; t1/2) was used to compare the stability of the different biocatalysts, being
determined by interpolation from the respective models described by Eq. (1) or Eq.
(2).
2.2. Methods
2.2.6. Oxidation of different compounds
In all cases the experiments were performed in triplicate and the maximal error
was never higher than 5%.
Commercial enzyme (0.02 mg) or 0.04 g of the most stable immobilized deriva-
tive (5 mg of enzyme per gram of support) was added to 1 mL of a solution at pH 7
or 4.5 of different compounds (2,6-dichloro-indophenol, pyrogallol and catechol).
In order to measure the activity with the different substrates, derivatives with
low load of enzyme were used again. In all cases activity was expressed as ␮mol of
product min−1 mg−1 of immobilized enzyme. At different times, aliquots of solution
were taken and the absorbance measured. Supports were previously saturated with
different substrates to avoid possible absorptions. Molar extinction coefficients (ε)
were: 12,097 M−1 cm−1 at pH 7 measured at 600 nm and 12,109 M−1 cm−1 at pH
4.5 at 520 nm for 2,6-dichloro-indophenol; 928 M−1 cm−1 (at pH 7 and 4.5) mea-
sured at 450 nm for o-benzoquinone, oxidation product from catechol; and finally
4283 and 3724 M−1 cm−1 at pH 7 and 4.5, respectively, and measured at 420 nm
for purpurogallin that is the oxidation product of pyrogallol as previously described
[43,44].
2.2.1. Enzyme assay
Standard laccase activity was determined by oxidation of ABTS at 25 ◦C [39].
The substrate solution was composed by 2 mL of ABTS (3 mM) in sodium acetate
buffer (10 mM at pH 4.5). Then a suitable amount of soluble enzyme or immobilized
preparation was added and the oxidation of ABTS was followed by measuring the
increase of the absorbance at 418 nm in a 1 cm path length spectrophotometric cell
(εABTS = 36,000 M−1 cm−1). One international unit (IU) of laccase activity corresponds
to the oxidation of 1 ␮mol ABTS per minute under these conditions.
2.2.2. Chemical amination of laccase
A mixture composed by a T. versicolor laccase solution (1 mL with 1 IU mL−1),
5 mL of 1 M ethylene diamine (EDA) at pH 4.75 and 0.012 g of solid 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDAC) was performed. After 90 min of gentle
stirring at 4 ◦C, the solution was dialyzed 5 times against 50 volumes of distilled
water and stored at 4 ◦C [40].
2.2.7. Reuse of the derivatives
In order to assess the operational stability of the most stable preparations, sev-
eral consecutive cycles were performed measuring the increase in the absorbance
using ABTS (3 mM) as substrate in sodium acetate buffer (10 mM at pH 4.5) at 418 nm
and 25 ◦ C. After each cycle, the immobilized preparation (20 IU/mL) was washed
three times with 5 volumes of acetate buffer (10 mM at pH 4.5).
2.2.3. Preparation of the supports
Cyanogen bromide Sepharose was hydrated as suggested by the supplier (1 g dry
support was suspended in 10 mL of water at pH 2–3 and stirred for 30 min. Then, sup-
port was dried under vacuum before being used for the immobilization). Sepharose
aldehyde supports were activated and oxidized as was previously described [41].
2.2.8. Determination of the enzyme structure
Enzyme structure was extracted from Protein Data Bank (PDB file: 1gyc). Model-
ing was performed using the program Pymol Molecular Graphics System. Amination
of the structure was simulated using this program considering a total amination of
carboxylic moieties.
2.2.4. Immobilization of the enzyme
A laccase solution (9 mL with 1 IU mL−1 or the correspondent amount in the max-
imal load of enzyme experiments) in sodium bicarbonate (0.1 M) at the indicated pH
(9 or 10) was added to 1 g of aldehyde-Sepharose. At different times, samples of the
supernatant and suspensions were taken and the activity was assayed. When the
immobilization process was completed, the different preparations were incubated
at the indicated pH (9 or 10) during 3 h. Then, 10 mg of solid NaBH4 were added
in a single addition and left under stirring during 30 min. Finally the support was
washed with water.
3. Results and discussion
3.1. Immobilization of the enzyme on aldehyde supports
Immobilization yield was referred to the percentage of enzyme activity that
disappeared from the enzyme solution compared with the activity of a blank solution
without support.
Expressed activity was calculated as the ratio between the final activity after
incubation at different pHs of the immobilized enzyme and the immobilized enzyme
activity measured at the optimal pH. All experiments were performed using a low
As it was commented above, the availability of a heterogeneous
and strongly stable biocatalyst is key for use at industrial scale.
Thus, laccase was offered to an aldehyde support for its immobi-
lization. The immobilization on aldehyde supports is performed by
a multipoint mechanism [41]. Therefore, incubation at alkaline pH
Please cite this article in press as: Addorisio V, et al. Oxidation of phenyl compounds using strongly stable immobilized-stabilized laccase from
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Fig. 1. Study of the structure of laccase from T. versicolor. Images were generated using Pymol Molecular Graphics System. Blue regions represent the primary amino groups.
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
is necessary in order to have the lysines unprotonated and conse-
quently in reactive form. After incubation of the laccase with the
aldehyde support at pH 10 during 24 h, the immobilization resulted
negligible. This fact indicates that the enzyme does not have an area
rich in lysine. This is fairly common when dealing enzymes from
fungi. This was confirmed analyzing the sequence of the protein
where it is seen that consists of only 8 lysines in all its secondary
structure. Despite having a zone of its surface with several lysines,
this seems not to be sufficient to promote the immobilization of
the enzyme (Fig. 1) [21]. Although the enzyme has 8 glycoxila-
tion points located mostly on one side. This may make difficult the
immobilization of the enzyme through this area.
into amine groups. Thus the enzyme would be strongly enriched
in amine groups with lower pKa than that of lysine. Once again the
number of acidic aminoacids was studied. This laccase has 29 aspar-
tics and 8 glutamics that could be potentially aminated using this
methodology. Identically, the 3D structure was studied and there is
a high superficial density of acid aminoacids. In Fig. 2, a model of the
possibly obtained enzyme is shown. This new protein would have
some places with higher density of these strongly reactive amine
groups.
The activity of the enzyme after the amination was approx-
imately 90% compared with the unmodified enzyme. This fact
confirms that the amination of acid groups was not complete, tak-
ing into account that there is an aspartic moiety key for the catalytic
activity of the enzyme.
One possible solution to this problem consists in the enrich-
ment of the enzyme in amine groups. The chemical amination of
the enzyme using ethylendiamine allows the conversion of the
carboxyl group of acidic aminoacids (glutamic and aspartic acid)
The aminated protein was offered to the aldehyde supports for
its immobilization at different pHs. The immobilization at pH 9
Fig. 2. Theoretical structure of aminated laccase from T. versicolor. Images were generated using Pymol Molecular Graphics System. Blue regions represent the primary amino
groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Please cite this article in press as: Addorisio V, et al. Oxidation of phenyl compounds using strongly stable immobilized-stabilized laccase from
Trametes versicolor. Process Biochem (2013), http://dx.doi.org/10.1016/j.procbio.2013.05.021

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Table 1
Immobilization yield and expressed activity of laccase derivatives. 100% corresponds to 9 IU g−1 support.
Laccase derivative
Immobilization yield (%)
Expressed activity (IU g−1
)
Expressed activity (%)
CNBr-Sepharose
46
<5
87
80
80
3.9
ND
4.3
6.6
5.8
93
Laccase-aldehyde pH 10
Laccase-NH2-aldehyde pH 10
Laccase-NH2-aldehyde pH 9
Laccase-NH2-aldehyde pH 9, 10
ND
59.6
92.2
74
ND: not determined.
must be promoted through the region of the protein richest in new
amine groups and the immobilization at pH 10 have to be produced
by the richest place in total amine groups (lysines and the new
obtained amine groups). The aminated enzyme was immobilized
in less than 1 h not only at pH 10 but also at pH 9 confirming
that the new amine groups had lower pKa than that of the lysines
and consequently they were more reactive. The activity recovered
just after immobilization on aldehyde supports was around 90%
of the pre-immobilized activity regardless of the immobilization
conditions showing that there are no diffusion problems with
this enzyme load. Then, different derivatives were incubated at
the indicated pH during 3 h in order to increase the multipoint
covalent linkage degree. The activity of the different derivatives
was 74% when the enzyme was immobilized and incubated at pH
10 and over 90% after immobilization and incubation at pH 9. A
decrease occurred after incubating this derivative immobilized
at pH 9 and at pH 10 with a final recovered activity of 74% and
60%, respectively (Table 1). The maximal obtained values (92%)
were similar to the best described in the literature using other
low distorting methodologies. Controlled porosity carrier silica
beads activated with glutaraldehyde allow catalysts where the
activity was high and similar to this study depending on the used
sustrate [45]. Cabana et al. [19], obtained activities similar to that
of the soluble enzyme after immobilization on chitosan supports
cross-linked by a polymer. Bayramoglu et al. [36] obtained 90%
recovery after immobilization on magnetic chitosan beads.
3.2. Study of the stability of the different preparations
The different derivatives were incubated in inactivating con-
ditions such as high temperature, different pH or in strongly
deleterious organic co-solvents as dioxane. The temperature of
inactivation for different conditions was adequate in order to pro-
mote the inactivation of the different preparations in a reasonable
time of performing the experiments. In all cases, the preparations
of enzyme immobilized on aldehyde supports were more stable
than native soluble enzyme or the soluble aminated enzyme. The
most stable derivative was that immobilized on aldehyde supports
at pH 10 and incubated in this condition during 3 h. Longer incuba-
tion times did not yield more stable derivatives (data not shown).
In addition, the derivative immobilized at pH 9 and incubated at
pH 10 was more stable than that immobilized at pH 9 but not incu-
bated (Fig. 3). This is related to the increase in the multi-interaction
enzyme-support promoted at pH 10. Taking into account that the
final step performing these two derivatives is the incubation at pH
10, the fact that the derivative directly immobilized at pH 10 is
100
100
B
A
75
50
25
0
75
50
25
0
0
10
20
30
40
50
60
0
10
20
30
40
50
Time, (h)
Time, (h)
100
75
50
25
0
D
100
75
50
25
0
C
0
10
20
30
40
50
60
0
10
20
30
Time, (h)
Time, (h)
Fig. 3. Stability studies of the different preparations after incubation in different conditions. Experiments were performed by measuring the activity at different times after
incubation in different conditions. Different preparations were diluted 10 times with the selected buffer or solvent. 100% was considered the initial activity of the different
preparations. (ꢀ) soluble laccase; (ꢁ) soluble laccase aminated with EDA; (ꢁ) laccase immobilized on CNBr-Sepharose and aminated with EDA; (ꢂ) laccase immobilized on
CNBr-Sepharose (open circle and square symbols were only applied to panel D (dioxane)); (᭹) laccase just immobilized on aldehyde supports at pH 9 and then incubated
at pH 10; (ꢂ) laccase immobilized at pH 10; (ꢃ) laccase immobilized at pH 9. (A) Incubated in 50 mM sodium phosphate at pH 7 and 55 ◦C; (B) incubated in 50 mM sodium
acetate at pH 5 and 50 ◦C; (C) incubated in 50 mM sodium bicarbonate at pH 9 and 40 ◦C; (D) incubated in 70% dioxane at 4 ◦C and pH 7.
Please cite this article in press as: Addorisio V, et al. Oxidation of phenyl compounds using strongly stable immobilized-stabilized laccase from
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Table 2
Stabilization of the different aldehyde derivatives. Different derivatives were incubated after dilution 1:10 with the indicated buffer. Reference preparations were soluble
and soluble-aminated enzyme or the enzyme immobilized on cyanogen-bromide support and this aminated derivative.
Stabilization factora
Incubation conditions
pH 7at 55 ◦
C
pH 5 at 50 ◦
C
pH 9 at 40 ◦
C
Dioxane 70%
Glyoxyl pH 9
7
19
8
12
9
32
1.25
1
Laccase preparation
Glyoxyl pH 9,10
Glyoxyl pH 10
15
103
Soluble
40
280
NH2− soluble
12
16
Soluble
17
23
13
16
Soluble
45
56
3
5
2.5
4.5
Cyanogen
bromide
aminated
Reference preparation
NH2− soluble
NH2− soluble
Cyanogen
bromide
a
Stabilization factor was calculated as the ratio between the half-life of different immobilized preparations and the reference preparations. In the presence of solvent
laccase immobilized on CNBr-Sepharose and the aminated derivative were used as reference of soluble and aminated soluble enzyme, respectively.
more stable than that immobilized at pH 9 and incubated at pH 10
suggests that the orientation of the enzyme may be different.
When the different preparations were incubated at pH 7 and
55 ◦C the derivative immobilized at pH 10 retained approximately
50% of its catalytic activity after 54 h, while soluble enzymes
(aminated and wild enzyme) had a half-life around 0.5 and 0.2,
respectively. The stabilization factors of this aldehyde derivative
were 103 and 280 if compare with native and aminated soluble
enzyme, respectively. Although laccase was stabilized using dif-
ferent methods the stabilization factor compared with the soluble
enzyme is not commonly reported. Due to this, comparison with
literature is quite difficult. Similar results were obtained after incu-
bation at acid and basic pH (Fig. 3). When different preparations
were incubated in medium with high concentration of organic
co-solvent (70% v/v dioxane) the aggregation and precipitation
of soluble enzyme occurred. The precipitation of the enzyme is
described that many times promotes its stabilization [46]. Thus,
in order to compare the enzyme in these conditions, a reference
one-point covalent immobilized derivative was used. These deriva-
tives have in general similar properties compared with soluble
enzyme [47]. In this case, the enzyme immobilized on commer-
cial cyanogen bromide Sepharose for a short time was used. This
derivative had similar stability to the soluble enzyme when it was
incubated at high temperature and in the absence of solvent (data
not shown). In addition, the derivative immobilized on cyanogen
bromide Sepharose was aminated as was described for the solu-
ble enzyme. This derivative was used as reference instead of the
aminated soluble enzyme. The general trend in the stability of
the different derivatives was similar to that obtained in the other
analyzed conditions but in this case the stabilization factors were
lower. The different obtained stabilization factors are summarized
in Table 2.
expected (around 90%). Other reported loads are 420 mg/g sup-
port when the enzyme was immobilized on carbon nanotubes [31]
or 56.4 after immobilization on poly(4-VP) grafted and/or Cu(II)
chelated magnetic beads via adsorption [36].
3.4. Reuse of the catalyst
The reuse of the biocatalyst is an important parameter in order
to evaluate the possible implementation of the catalyst at indus-
trial level. The use of the catalyst in successive reaction cycles was
checked using ABTS as substrate. The most stable catalyst could be
used during at least 10 reaction cycles with negligible changes in
the activity (Fig. 5A). This catalyst was also used in the oxidation
of cathecol. After 5 reaction cycles the activity remained unaltered
(Fig. 5B).
3.5. Oxidation of different phenolic compounds
The native soluble enzyme and the most stable derivative were
used for the oxidation of various phenolic compounds at different
pHs, the optimal of the enzyme (4.5) and neutral pH. The results
are summarized in Table 3 where the initial enzymatic activity is
presented. As it can be observed, in all cases the activities were
higher at pH 4.5 than at pH 7 for soluble enzyme as well as for the
derivative immobilized at pH 10. The highest activities were found
with catechol and pyrogallol. The yields of the reaction were the
obtained using soluble enzyme as it was expected. For example,
using cathecol, 100% of conversion was found. This result agrees
with that obtained by Canfora et al. [48]. This demonstrated the
high applicability of the different preparations that is expected to
The stabilization factor of the most stable derivative is the high-
est published until the knowledge of the authors. This fact may
convert the catalyst in a useful tool in different decontamination
processes.
70
60
50
40
30
20
10
0
3.3. Maximal load of the supports
The industrial catalysts have to be loaded maximizing the activ-
ity in order to add the lowest possible amount of the catalyst to
the industrial reaction. For calculating the maximal load of the
support different amounts of enzyme were added to the support.
The maximal amount of enzyme immobilized was approximately
60 mg g−1 of support (Fig. 4). In the case of the most stable deriva-
tive the measured activity was the maximal expected using ABTS
as substrate. This is a typical result taking into account that dur-
ing the stabilization process the activity decreased until 60%. In the
case of the derivative immobilized at pH 9 the activity measured
was around 75% of the expected (92.2%) (Table 1). This decrease of
the activity was promoted by diffusion limitations. Using other of
the assayed phenolic compounds the recovered activity was that
0
20
40
60
80
100
Offered laccase (mg)
Fig. 4. Maximal load of laccase on Sepharose-6BCL aldehyde supports. The load of
enzyme is referred to 1 g of support.
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100
75
50
25
0
100
75
50
25
B
A
0
1
2
3
4
5
1
2
3
4
5
6
7
8
9
10
Cycle number
Cycle number
Fig. 5. Reusability of the most stable Sepharose-aldehyde derivative in the oxidation of ABTS and cathecol. Cathecol concentration was 1 mM in sodium acetate 100 mM at
pH 4.5.
Table 3
Oxidation of different phenolic substrates.
Substrate
Soluble enzyme (␮mol/min/mg protein)
Aldehyde derivative (␮mol/min/mg protein)
pH 7
pH 4.5
pH 7
pH 4.5
2,6-Dichloro-indophenol
Pyrogallol
Catechol
0.028 0.0014
0.073 0.00365
0.034 0.0017
0.057 0.0285
0.120 0.006
0.104 0.0052
0.015 0.00075
0.044 0.0022
0.022 0.0011
0.034 0.0017
0.078 0.0039
0.069 0.00345
be useful for the oxidation of other different phenolic compounds
as well as for other different compounds.
[4] Strong PJ, Laccase Claus H. A review of its past and its future in bioremediation.
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4. Conclusions
Laccase from T. versicolor was used as immobilized catalyst to
oxidize different phenolic compounds. A key point for this is the
availability of highly active and stable biocatalyst. Laccase was
chemically aminated to be immobilized on aldehyde Sepharose
supports. Immobilized derivatives had a high activity (over 60%)
compared with soluble enzyme. Best derivatives were that directly
immobilized at pH 10 and incubated during 3 h. They resulted
more stable than soluble enzyme after incubation in inactivating
conditions (high temperature, extreme pHs or presence of organic
solvents). The immobilization on this support can reach 60 mg of
laccase g−1 of support. The most stable derivative could be reused
for at least 10 reaction cycles with negligible lost of activity.
Although the immobilization of this laccase has been immo-
bilized by different techniques, catalytic activity per mass unit,
stabilization factors or mass load capacity is not commonly
reported. There are reports that characterize one of these parame-
ters but a global comparison in terms of activity, stability and load
capacity of different biocatalysts is difficult to be done.
[14] Jeon JR, Kim EJ, Murugesan K, Park HK, Kim YM, Kwon JH, et al. Laccase-
catalysed polymeric dye synthesis from plant-derived phenols for potential
application in hair dyeing: enzymatic colourations driven by homo- or hetero-
polymer synthesis. Microb Biotechnol 2010;3:324–35.
[15] Cerrone F, Barghini P, Pesciaroli C, Fenice M. Efficient removal of pollutants from
olive washing wastewater in bubble-column bioreactor by Trametes versicolor.
Chemosphere 2011;84:254–9.
The obtained results convert this derivative in a promising tool
in the oxidation of different phenyl compounds at industrial scale.
[16] Majeau JA, Brar SK, Tyagi RD. Laccases for removal of recalcitrant and emerging
pollutants. Bioresour Technol 2010;101:2331–50.
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Trametes versicolor to chitosan and its utilization for the elimination of triclosan.
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Acknowledgments
This work has been sponsored by the Spanish Ministry of Science
intramural projects (2008801058).
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