Environment Protection Engineering Vol. 42 2016 No. 2 DOI: 10.5277/ epe160205 MIHAELA MURE ȘEANU1, ION TRANDAFIR1, CRISTINA BĂBEANU1, VIORICA… [602477]

Environment Protection Engineering
Vol. 42 2016 No. 2
DOI: 10.5277/ epe160205

MIHAELA MURE ȘEANU1, ION TRANDAFIR1, CRISTINA BĂBEANU1,
VIORICA PÂRVULESCU2, GABRIELA PĂUN3
LACCASE IMMOBILIZED ON MESOPOROUS
SILICA SUPPORTS AS A N EFFICIENT SYSTEM
FOR WASTEWATER BIOREMEDIATION
The feasibility of using laccase from Trametes versicolor for degradation of aromatic hydrocar-
bons has been investigated . In the experiments, b enzo[a]pyrene (BaP) was used. Laccase was immobi-
lized onto mesoporous micelle -templated silica such as Santa Barbara Amorphous ( SBA -15) and hex-
agonal mesoporous silica ( HMS ) as well as corresponding amino -functionalized supports . The b est
results were obtained for SBA -15 mesoporous silica however the HMS support could be as well con-
sidered for this type of application . The reusability of laccase immobilized into both silica supports
was tested for five reaction cycles and the conversion reached about 70% of the initial value.
1. INTRODUCTION
This work is a part of a study whose ultimate goal is to propose a complete
wastewater bioremediation treatment for removal of toxic compounds (e.g. polycyclic
aromatic hydrocarbons , phenols, organophosphorous, pesticides, pigments) and of
heavy metals with coupling phenol oxidase enzymes and some selective biolo gical che-
lators anchored at the surface of mesoporous silica supports and packed into fixed bed
columns. A mesopor ous hybrid silica bio -adsorbent based on a low-molecular weight
protein metallothioneine was already tested for selective adsorption of Cu(II) ions from
contaminated water bot h in batch and in -flow process [1].
_________________________
1Faculty of Mathematics and Natural Sciences, Department of Chemistry, University of Craiova, Cra-
iova, Romania , corresponding author M. Mureșeanu, e -mail: [anonimizat]
2Institute of Physical Chemistry, Romanian Academy of Science s, Bucharest, Romania .
3National Institute of Research and Development for Biological Sciences, Center of Bioanalysis, Bu-
charest, Romania .

82 M. MUREȘEANU et al.
Phenol oxidases are enzymes that catalyze reaction of dioxygen reduction to water
with production of substrate radicals which are non -enzymatically converted to dim-
mers, oligomers and polymers [2]. Increasing numbers of oxidative biotransformation
of phenol oxidases, especially laccases (EC 1.10.3.2), have been extensively reviewed
[3–5]. Laccases can be involved in the detoxification of phenols, trichlorophenols,
organophosphorous pest icides, azo dyes and, interestingly, PAHs, a class of highly
mutagenic and carcinogenic xenobiotics, widely distributed in terrestrial and aquatic
environments [6 –10].
Thus, using enzymes as decontaminating agents has received great attentions be-
cause of their potential to remove pollutants from environment without the harsh side
effects associated with many other methods. However, operational stability of native
enzymes is rather limited. Therefore, immobilization is very often applied in order to
overcom e this limitation, as was summarized by Fernández -Fernández et al. [11] al-
lowing one to develop many possible applications, including bioremediation, chemi-
cal synthesis, biobleaching of paper pulp, biosensing, textile finishing and wine sta-
bilization.
Laccases have been successfully immobilized on many different types of carriers
such as magnetic chitosan microspheres [12], nanoparticles and kaolinite [7], meso –
structured siliceous cellular foams [13], mesoporous silica [14], fibrous membrane s
[15] or tit ania nanoparticles and titania -functionalized membranes [16]. Among these,
the mesostructured sol -gel materials which possess highly ordered structure, with pore
sizes similar or larger than most enzyme molecules, showing very high chemical, ther-
mal, mecha nical and biological resistance afford preparations with ultrahigh activity
[13, 17]. Mesoporous micelle -templated silica such as Santa Barbara Amorphous
(SBA -15) and hexagonal mesoporous silica (HMS) have currently been receiving
great attention due to th eir unique properties of highly controlled and uniform pore
size and high values of surface area and pore volume. Despite of the HMS being less
ordered than the MCM -41 and SBA -15 materials, they are much easier and straight-
forward to synthesize due to the use of a cheap and neutral template, i.e. long chain
amines, which does not require strongly acidic conditions.
In the present work, laccase from Trametes versicolor was immobilized on SBA -15
and HMS type mesoporous silica and on NH 2-functionalized mesop orous silica by
either physical adsorption or covalent binding when the supports were activated by
cross -linking with glutaraldehyde. To the best of our knowledge, immobilization of
laccases has not been carried out yet on HMS mesoporous silica support. Th e effi-
ciencies of these immobilized laccases on degradation of BaP in the presence and in
the absence of redox mediators as well as the reusability of the catalyst were presented
in order to develop detoxification processes of aromatic compounds from conta mi-
nated waters.

Laccase immobilized on mesoporous silica supports 83
2. EXPERIMENTAL
Materials. Benzo[a]pyrene (BaP), 2,2-azino -bis(3 -ethylbenzothiazoline -6-sulfonic
acid) (ABTS), acetone and acetonitrile were obtained from Sigma. Laccase (E.C.1.10.3.2)
from Trametes versicolor was also purchased from Sigma with an activity of 33 I.U./mg
protein and used without further purification. Poly(ethylene oxide) -poly(propylene oxide) –
-poly(ethylene oxide) (EO 20PO 70EO 20), dodecyl amine (DDA), and tetraethyl orthosili-
cate (TEOS, 98%) wer e obtained from Aldrich .
Synthesis of functionalized silica . SBA -15 material was synthesized as described by
Zhao et al. [18], while HMS silica was prepared according to the procedure described
by Tanev and Pinnavaia [19]. The organic -inorganic hybrid mater ials were obtained by
a post -grafting procedure with 3 -aminopropyl -triethoxysilane (APTES) [20]. The NH 2-
-functionalized mesoporous silica were referred as SBA -15-NH 2 and HMS -NH 2, respec-
tively .
Immobilization of laccase into mesoporous silica supports . Immobilization of lac-
case was carried out by either physical adsorption or covalent coupling. In a typical
procedure for physical adsorption, 1 g of support (SBA -15, SBA -15-NH 2, HMS, or
HMS -NH 2) was mixed with 10 cm3 of 0.1 M phosphate buffer (pH 7.0) in a centrifuge
tube. Thereafter, 50 mg laccase in 3 cm3 phosphate buffer were added to the mixture
and homogenized under magnetic stirring at 278 K. Acetone (30 cm3) was then added
drop wise and the stirring was continued for another 30 min. The solids were i solated
by centrifugation and washed several times with buffer until no laccase activity was
detected in the washing. The immobilized systems were then lyophilized and stored in
dark at 253 K.
In the case of covalent binding , the procedure involves the mi xing of 1g SBA -15-
-NH 2 with 25 cm3 of 5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7) for
30 min. Then, the excess of glutaraldehyde was removed during three cycles of centrif-
ugation/washing with 10 cm3 buffer solution each. Enzyme (50 mg ) in 13 cm3 of phos-
phate buffer (pH 7.0) was added to the activated solid. The suspension was stirred at
278 K for 24 h, centrifuged at 3000 rpm for 10 min to remove the buffer, and washed
several times with buffer until no laccase activity was detected in the wa shing. The solid
thus obtained was lyophilized and stored at 253 K (sample denoted SBA -15-NH 2 cov).
The concentration of p rotein in the supernatant was determined by the Bradford
procedure [21] using bovine serum albumin as a standard . The amounts of immo bilized
laccase were evaluated by the thermogravimetric (TG/DTG) method and compared with
the results of the Bradford assay.
Characterization of solids . Small -angle X-ray diffraction (XRD ) data were acquired
on a Bruker diffractometer using Cu Kα radiation. N 2 adsorption -desorption isotherms

84 M. MUREȘEANU et al.
were recorded at 77 K with a Micromeritics ASAP 2010 instrument. The samples were
degassed at 323 K for 12 h prior to the adsorption measurements. Spec ific surface area
was calculated by the Brunauer, Emmett and Teller (BET ) theory, the mesopore volume
was determined by nitrogen adsorption at the end of capillary condensation, and pore
size distribution was determined from the desorption branches of the isotherms by
Broekhoff and de Boer (BdB) theory . Thermogravimetric analysis was carried out using
a Netzsch TG 209C thermobalance.
Measurements of the s pecific activity and estimation of kinetic parameters . The ac-
tivities of free and immobilized laccase w ere determined spectrophotomet rically by the
rate of oxidation of 1 mM ABTS in 0.1 M acetate buffer (pH 4.5) at 29 8 K. The substrate
solution was aerated by air bubbling before the addition of 0.01 cm3 of 5 mg cm–3 en-
zyme solution for 3 cm3 reaction mixtur e. During the process, the increase in absorbance
at 424 nm was measured ( ε = 36 000 dm3·mol–1·cm–1 for the oxidation product of
ABTS). International unit (IU) of laccase activity was defined as the amount of enzyme
required to oxidize 1 μmol ABTS per minute.
For the determination of the activity of immobilized laccase, 10 mg of active solids
were added to 50 cm3 of 1 mM ABTS in 0.1 M acetate buffer (pH 4.5), which was
allowed to circulate through the spectrophotometric cell at 29 8 K. The acti vities of the
immobilized laccase during the process were determined from the plot of absorbance
versus reaction time at 424 nm . The enzymatic activity was expressed in IU/mg of im-
mobilized protein or as relative activity with respect to the activity of th e free enzyme .
Kinetic parameters of free and immobilized laccases were determined by using
ABTS as substrate in the concentration range of 0.05 –2 mM. The Michaelis –Menten
kinetic parameters were obtained by the nonlinear regression analysis using Origin 6 .0
from the plot of the initial reaction rates versus concentration of substrate, based on the
following equation:


max cat 0S [S][]S [S]mmV k EKK (1)
where  (mM ·min–1) is the reaction rate and [S] is the substrate concentration.
Vmax (mM ·min–1) is the maximum rate achieved by the biocatalyst. The Michaelis con-
stant Km (mM) is the substrate concentration at which the reaction rate is half of Vmax.
[E] 0 is the enzyme concentration and the reaction rate constant kcat (mM ·s–1·g–1) is the
maximum number of substrate molecules converted to product per enzyme molecule
per second.
All assays were replicated three times. Control samples contained boiled immobi-
lized laccase were used to determine the amount of ABTS absorbed on or react ed with
the support.

Laccase immobilized on mesoporous silica supports 85
Properties of immobilized laccase . Thermal stability of the immobilized enzyme
was evaluated by measuring the remaining activities after sample incubation in acetate
buffer (pH 4.5) for 1 h at temperature ranging from 313 K to 353 K.
The pH -stability was determined by incubating the samples for 1 h at 298 K in buff-
ers of varying pH in the range of 2.0–10.0. pH of the solution was then adjusted to 4.5
and its activity was measured after equilibration. The residual activity was expressed as
a percentage relative to the initial enzyme activity.
Oxidation efficiency of immobilized laccase on BaP . The oxidation treatments were
performed in 20 cm3 reaction volumes in amber bottles containing: phosphate buffer
(pH 7), BaP dissolved in acetone to make the final concentration 20 μM, free or immo-
bilized laccase adjusted to 4 IU/cm3. The impact of ABTS on oxidation of BaP was
determined by its addition to some samples to a final concentration of 1.0 mM. The
reaction mixture was aerated by air bubbling before the enzyme addition. The reaction
bottles were incubated in an orbital shaker at 150 rpm and 318 K . The reaction mixture
was then centrifuged at 4000 rpm for 10 min, the supernatant was decanted and then the
same volume of acetonitrile was added to inactivate the enzyme and shaken for half an
hour to extract the BaP. Control sample for the free laccase was prepared in the same
manner but the enzyme was inactivated by boiling for 1 h. For the immobilized enzyme
system, the controls contai n boiled immobilized laccase onto solid supports for evalu-
ating the sorption of BaP by the supports. The percentage of aerobically oxidized BaP
was calculated from the difference between the BaP levels in the experimental assay
and the corresponding contro l. All treatments, including controls, were replicated three
times. The biocatalyst recycling was also investigated for the laccase immobilized by
physical adsorption into HMS -NH 2 support or by covalent coupling onto SBA -15-NH 2
by using the same amounts of catalyst and substrate and the previously described pro-
cedure.
The separation and quantification of BaP were achieved by using a Surveyor
Thermo Electron HPLC system (Thermo Scientific) comprising a vacuum degasser,
Surveyor Plus LCPMPP pump, a Surveyor P lus ASP autosampler, a diode array detector
with a 5 cm flow cell and a Chrom Quest 4.2 software. The Hypersil G reen PAH column
with 5 m packing (250 mm  4.6 mm i.d.) was used in the analysis. The determinations
were made in isocratic conditions, at 283 K using a mixture of acetonitrile/water
(80:20 v/v) as the mobile phase . The volume injected was 5 ·10–3 cm3 and the flow rate
of the mobile phase was 1 cm3·min–1. The wavelength was set at 275 nm to integrate the
peaks. A good resolution was obtained for BaP (2.75 min). The BaP conversion was
calculated as the ratio between the amount of oxidized BaP and the corresponding con-
trol. The c alibration curves were constructed in the range of 1 –200 ppb. Linearity was
achieved and the correlation coefficient was 0 .999. All analys es were performed in trip-
licate with reproducibility always within 3%.

86 M. MUREȘEANU et al.
Samples of the reaction solution were extracted with n-hexane. The extracts were
analyzed by the CG/MS to identify the oxidation products of BaP. Analysis was per-
formed using a GC/MS (DSQ II Thermo Scientific) equipped with a Thermo TR -5MS
(30 m × 0.25 ID × 0.25 μm film thickness) capillary column. The GC carrier gas was
helium, at the flow rate of 1 cm3·min–1. The GC temperature program was as follows:
313 K for 3 min , temperature ramp of 10 °C/min to 423 K and temperature ramp of
15 °C/min to 523 K , 523 K for 2 min.
3. RESULTS AND DISCU SSION
3.1. SYNTHESIS AND C HARACTERIZATION OF M ESOPOROUS MATERIAL S
SBA -15 and HMS mesoporous silica samples have been synthesised and the amino
moiety was grafted thereafter on the support surface by a post -synthesis procedure, due
to the interaction of surface hydroxyl groups with the ethoxy groups of APTES. The
densit ies of the functional groups grafted on the silica surface measured by thermo –
gravimetric analysis were 3.1 molecules/nm2 of pure SBA -15 silica and 4.2 mole-
cules/nm2 of HMS silica, respectively.

Fig. 1. Powder X -ray diffraction patterns of pure and amino -functionalized silica:
a) SBA -15, b) HMS
The XRD measurements confirmed the SBA -15 structure for both unmodified and
grafted samples (Fig. 1a). The SBA -15 material exhibited a strong (100) reflection peak
(at 2θ = 0.7) and smaller (110), (200), (210 ) diffraction peaks, which are characteristic
of a well ordered SBA -15 type material [18]. No significant changes upon amine im-
mobilization were observed, except for the expected decrease in XRD peak intensity,
providing evidence that functionalization occ urred mainly inside the mesopore chan-
nels.

Laccase immobilized on mesoporous silica supports 87
The XRD patterns of the functionalized HMS silica samples exhibit XRD reflec-
tions similar to that of the HMS unmodified support but intensit ies of the peaks decrease
significantly (Fig. 1b). The diffraction peak a t 2θ = 2.5 ° may be due to the d100 reflection
in materials with a short -range hexagonal order. Similar pattern have been reported for
ordered mesoporous materials such as HMS [22]. As already noted, these HMS materi-
als do not display the degree of long ran ge order associated with the MCM -41 class of
silicates.

Fig. 2. N2 adsorption -desorption isotherms of pure and amino -functionalized silica:
a) SBA -15, b) HMS
All SBA -15 type silica exhibited irreversible type IV adsorption -desorption iso-
therms (Fig. 2a) with a H1 hysteresis loop in the relative pressure range from 0.65 to
0.75, characteristic of materials with 7 –8 nm pore diameter. This result reveals that the
uniform mesoporous nature of the material is preserved even thoug h the grafting has
occurred. In Table 1 , the main textural properties of solids have been listed: specific
surface area SBET, mesopore volume Vmezo, and pore diameter DBdB calculated based on
the BdB theory .
T a b l e 1
Textural properties of mesoporous silica supports
Material SBET
[m2·g–1] Vmezo
[cm3·g–1] DBdB
[nm]
HMS
HMS -NH 2
SBA -15
SBA -15-NH 2 1029
510
697
368 0.79
0.30
1.49
0.77 3.5
2.3
8.3
7.8

For the functionalized SBA -15 materials, the BET surface and volume were stand-
ardized versus pure silica weights . As expected, the BET surface area and the mesopore

88 M. MUREȘEANU et al.
volume strongly decreased after grafting. The nitrogen adsorption isotherm at 77 K of
the calcined HMS material (Fig. 2b) is of type IV, featuring a sharp step corresponding
to the filling of the ordered mesopores in the p/p0 range of 0.1 –0.5 and a H1 hysteresis
loop at p/p0 of 0.5, being therefore typical of mesoporous materials. The surface area
decreased for the HMS -NH 2 sample (Table 1). These textural results confirm that the
grafted spe cies are located inside the mesopores and not only on the outer surface of the
mesoporous silica materials.
3.2. IMMOBILIZATION AND S PECIFIC ACTIVITY OF LACCASE
Enzyme immobilization is an effective approach to enhance the enzyme stability. It
is well-known that material morphology, surface chemistry, activation and immobiliza-
tion conditions have an important effect on the enzyme immobilization. Mesoporous
materials , ideal carriers for enzyme s due to their nanostructures , have been actively de-
velop ed to immobilize enzymes in the recent years [23]. Previous studies revealed that
by immobilization of laccase from T. versicolor on various supports an improvement of
its catal ytic properties such as pH and temperature , stability or reusability , compared to
those of the free laccase was obtained . Moreover, if the support is a highly ordered silica
material, a further improvement in the enzyme binding capability and a decrease of the
diffusional limitations, as well as a shift of the optimal pH towards neut rality were
achieved [11].
In this study Trametes versicolor laccase was immobilized by adsorption onto SBA -15,
HMS , and the corresponding amino -functionalized mesoporous silica in order to obtain
an efficient catalyst for the ABTS substrate oxidation. Fo r the SBA -15-NH 2 support, the
covalent coupling of laccase was also performed. The amount of immobilized enzyme
was determined either as equivalent bovine serum albumin by the Bradford assay or as
organic mass loss from TG/DTG curves (Table 2). The amount of glutaraldehyde used
as a cross -linking agent for the covalent grafting was previously determined from the
thermogravimetric analysis assuming total conversion of grafted amine functions and
subtracted from the total mass loss in order to obtain the amou nt of immobilized laccase.
In the case of silica of HMS type, the enzyme immobilization yields are lower than
for SBA -15 silica material due to their pore sizes into which the enzyme cannot be in-
corporated. Therefore, for these materials the immobilizatio n can only take place on the
external surface.
The higher yields obtained for the laccase immobilization into the amino -function-
alized silica are probably due to the stronger interactions betwe en the enzyme functional
groups (–NH 2, –COOH, –OH, –SH) and that of the support surface. Furthermore, the
higher value corresponding to the HMS -NH 2 compared to the SBA -15-NH 2 can be as-
signed to the higher density of amino groups (4.2 molecules/nm2 comparatively with
3.1 molecules/nm2 of pure SBA -15 silica) . For enz yme immobilization by physical ad-
sorption, acetone was added to a mixture of the solid support and the buffered enzyme

Laccase immobilized on mesoporous silica supports 89
solution in order to produce a forced diffusion of the enzyme through the hydrophilic sur-
faces. This method is more efficient than other adsorption methods due to a better control
of protein deposition or agglomeration on solid/liquid interface.
The immobilization yield s for laccase immobilization by covalent binding are
slightly higher than by physical adsorption (Table 2). These results a re in agreement
with th ose reported in literature reviews [6, 7].
T a b l e 2
Amount and enzymatic activity of immobilized laccase
onto various supports determined by two different techniques
Material Bound protein [mg/g solid ] Immobilization
efficiencya
[%] Enzymatic
activityb
[IU/mg protein ] Relative
activityc
[%] Bradford
method TG
SBA -15 29.73 29.11 59.47 19.90 60.30
SBA -15-NH 2 33.67 34.12 67.33 27.75 84.10
HMS 20.47 20.03 40.93 16.73 50.68
HMS -NH 2 40.93 41.08 81.86 23.27 70.45
SBA -15-NH 2 cov 37.73 41.90 75.47 28.83 87.35
aCalculated using the Bradford meth od to evaluate the protein load.
bDetermined through the ABTS assay.
cAssuming that the specific activity of the free laccase is 33 IU/mg pr otein .

In order to evaluate the efficiency of the immobilized laccase from T. versicolor
into different mesoporous silica with different techniques, the solids were tested in the
ABTS oxidation reaction. The enzymatic activity and relative activity of the immobi-
lized laccase are given in Table 2. The obtained values suggest that the laccase activity
strongly depends on the nature of the support surface and the protein loading of the
support. Thus, the amino -functionalized silica allows immobilizing a greater amount of
protein, and , consequently , the enzymatic activity is higher. Even if for the HMS -NH 2
the yield of immobilization of laccase is higher than that for the SBA -15-NH 2, the rela-
tive enzymatic activity decrease s in accordance with observation of Salis et al. [24] .
They suggested that at higher loadings than a maximum value, a decrease of the laccase
activity occurs. Nevertheless, the highest relative enzymatic activity was obtained fo r
the laccase covalently bound onto the SBA -15-NH 2 support. However, a stress exerted
on proteins during the covalent attachment could produce their partial inactivation.
Thus, about 87.35% of the bou nd enzyme expressed their activity after immobilization
(Table 2 ). For all samples, a decrease of the enzymatic activity compared to that of free
enzyme was evidenced. This behavior can be assigned to the change of catalytically
active conformation due to the enzyme interactions with the support surfaces. Mass
transfer limitation, unfavorable substrate partition, or restrained enzyme flexibility
could also affect the enzymatic activity [25].

90 M. MUREȘEANU et al.
The kinetic parameters of the Michaelis –Menten equation applied to the activity of
free and immobilized laccases with good relative activity as a function of ABTS con-
centration were determined and the results are presented in Table 3.
T a b l e 3
Michaelis –Menten kinetic parameters for free
and immobilized laccase into vari ous silica supports
Support Vmax
[mM·min–1] Km
[mM] R2
Free 0.108 0.282 0.992
SBA -15-NH 2 0.085 3.475 0.993
SBA -15-NH 2 cov 0.043 0.185 0.990
HMS -NH 2 0.092 2.367 0.995

Only for SBA -15-NH 2 cov sample Km was lower than for free laccase which indi-
cates a higher affinity for the substrate after immobilization. Nevertheless, this lower
Km value for covalent immobilized enzyme was counterbalanced by the lowest Vmax.
For the other samples, the Km values were higher and the Vmax values were smaller,
compared with that of the free enzyme. This findings prove that immobilization de-
crease d the affinity for the substrate but this negative effect was compensated by the
increased stability of the immobilized biocatalysts. The internal and external limita-
tions such as mass transfer resistance, limited accessibility of active sites after immo-
bilization, the conformational changes of the protein molecule and steric hindrance
are the major factors that could lead to an increase in Km and a decrease in Vmax [16].
Fig. 3 . Effect of temperature on the activity
of free and immobilized laccase
in various mesoporous silica supports
The effect of temperature on the relative activity of the immobilized enzyme was
examined (Fig. 3). The optimal temperature (i.e. the temperature that shows a maximum

Laccase immobilized on mesoporous silica supports 91
relative activity) for all the immobilized lac cases ranged between 318 and 323 K. Lac-
case immobilized on SBA -15 or HMS mesoporous silica is more stable toward heat
denaturation in the 313 –353 K temperature range. Thus, after 1 h of incubation at 323 K,
SBA -15-NH 2 cov retained almost 100% of its initial activity, whereas free laccase re-
tained ca. 45% (Fig. 3). All immobilized enzymes show ed an analogous behavior in the
investigated temperatu re range, after 1 h incubation. Free laccase is denaturated and lost
its activity after expo sure at above 323 K, while immobilized laccase into different sup-
ports retained between 40% and 55% of its initial activity at the same temperature. The
temperature required to inactivate laccase immobilized on mesoporous silica was higher
than 353 K.
Fig. 4 . Effect of pH on the activity of free
and immobilized laccase
in various mesoporous silica supports
Investigations of pH effect on activity of free and immob ilized laccase showed that
activity profile from the bound enzyme is shifted toward neutrality (Fig. 4). The most
favorable pH of all the immobilized laccase s was 5.5 –6. Incubation of the enzymes in
buffers of various pH indicated that immobilization impro ved stability of laccase over
a wide pH range (4–7) compared with the free counterpart. At acidic solution (pH 2.0),
free laccase almost lost its activity but immobilized laccase retain ed more than 35%.
A similar behavior was observed under alkaline condit ions, indicating that the resistance
of laccase to acid and basic denaturation was considerabl y increased after immobiliza-
tion. The stability of pH indicated that the mesoporous supports created a more favora-
ble environment for immobilized laccase, which c ould make this one more applicable
to industrial use.
3.3. OXIDATION EFFIC IENCY OF IMMOBILIZED LACCASE ON B aP
Previous studies [6, 7, 15] on the oxidation of BaP with free and immobilized lac-
case showed a considerable increase of the conversion after addition of ABTS to the
reaction mixture. The role of ABTS is to mediate the oxidation of BaP by laccase [6, 7].

92 M. MUREȘEANU et al.
In our study, the reaction s of oxidation of BaP were investigated using free and five
immobilized laccase s, in absence or presence of ABTS medi ator. The highest BaP con-
version among the immobilized laccases was obtained for the SBA -15-NH 2 cov sample
(Fig. 5). These results are in agreement with the above mentioned laccase activities for
the ABTS oxidation. The conversion yields of biocatalysts ba sed on HMS supports
(40% for HMS -NH 2 and 28% for HMS, respectively) were lower than those for SBA -15
(52% for SBA -15-NH 2 and 35% for SBA -15, respectively) but their values are however
considerable and correlated with the amount of immobilized protein.

Fig. 5 . Oxidation of benzo[a]pyrene with free and immobilized laccases ;
318 K , 50 rpm; pH 7 , concentration of BaP 20 μM
The metabolites of the oxidation of BaP were identified after their extraction from the
reaction mixture in n-hexane using GC/MS analysi s. After 48 h of oxidation, the BaP was
stoichiometrically converted to its quinone derivatives. The identified metabolites were:
1,6-benzo[a]pyrene quinone (1,6 -BaQ), 3, 6-benzo[a]pyrene quinone(3,6 -BaQ), 6,12-
-benzo[a]pyrene quinone (6,12 -BaQ). These me tabolites were also observed in a previous
study [7]. Further studies will be performed to establish the isomer rat io and the oxidation
mechanism.
Due to the possibility to recycle the immobilized enzyme, the area of their techno-
logical applications can be extended. Therefore, the effect of its reuse as a catalyst in
several oxidation of BaP reaction cycles was investigated. The HMS -NH 2 and SBA -15-
-NH 2 cov samples were used as biocatalyst. The BaP conversion values after repeated
use of immobilized laccase in the oxidation of BaP mediated by ABTS are shown in
Fig. 6. After the 5 th reaction cycle the conversion reached about 58% for the SBA -15-
NH 2 cov sample and 25% for HMS -NH 2 sample, respectively. This means that after the
fifth cycle, the oxidative activity of the biocatalysts comparatively with their initial ac-
tivity was 85% for SBA -15-NH 2 cov and 63% for HMS -NH 2 sample, respectively. It is
clear that the biocatalyst obtained by covalent coupling of laccase to the mesoporous

Laccase immobilized on mesoporous silica supports 93
support was more stable toward the leaching effect during the multiple recycling. We
intend to immobilize laccase by covalent coupling into the HMS -NH 2 support, in order
to further com pare the reusability of laccase immobilized into the same support but by
different methods.

Fig. 6 . Effect of recycling immobilized laccase on oxidation of BaP : a) SBA -15-NH 2 cov,
b) HMS -NH 2, 318 K , 50 rpm, pH 7 , concentration of BaP 20 μM
4. CONCLUSIONS
Laccase from Trametes versicolor immobilized on ordered HMS or SBA -15 meso-
porous silica supports by physical adsorption or chemical bonding could be used for
effective PAHs remediation. The HMS silica support presented smaller , however con-
sider able activities for the ABTS oxidati on as well as for the conversion of benzo[a] py-
rene into oxidation products than SBA -15 silica . Considering that this support could be
easily synthesized from cheaper reagents, it could be used as an efficient material fo r
laccase immobilization and its application in biodegradation processes. This highly or-
dered inorganic mesoporous supports exhibit several benefits such as: the reproducibil-
ity of the material through a standardized synthesis procedure, easiness of their surface
modification in order to allow the enzyme immobilization, and high stability and reus-
ability of the obtained biocatalysts.
ACKNOWLEDGMENT S
This work was partially supported by the grant number 1C /2014, awarded in the internal grant com-
petition of t he University of Craiova .

94 M. MUREȘEANU et al.
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