Vol.3, No.1, 81-87 (2012) Journ al of Biophysical Chemistry
http://dx.doi.org/10.4236/jbpc.2012.31010
Laccases stabilization with phosphatidylcholine
liposomes
Meritxell Martí1*, Andrea Zille2, Artur Cavaco-Paulo3, José Luís Parra1, Luisa Coderch1
1Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain;
*Corresponding Author:
2IBMC—Institute for Molecular and Cell Biology, Porto, Portugal
3Textile Engineering Department, University of Minho, Guimarães, Portugal
Received 13 October 2011; revised 24 November 2011; accepted 8 December 2011
ABSTRACT
In recent years, there has been an upsurge of
interest in enzyme treatment of textile fibres.
Enzymes are globular proteins whose catalytic
function is due to their three dimensional struc-
ture. For this reason, stability strategies make
use of compounds that avoid dismantling or
distorting protein 3D structures. This study is
concerned with the use of microencapsulation
techniques to optimize enzyme stabilization. La-
ccases were embedded in phophatidylcholine
liposomes and their encapsulation capacity was
assessed. Their enzymatic activity and stability
were analyzed, comparing free-enzymes, enzy-
mes in liposomes, and the lipid fraction sepa-
rated from the aqueous fraction. An increase in
their encapsulation efficiency was found at
higher lipid/laccase ratios. Relative activity of
enzyme-containing vesicles has also been shown
to be ret ained muc h more than that o f free nativ e
enzymes. The loss of activity of laccases en-
trapped in the vesicles in the total stability pro-
cess is lower than 10% compared with 40% to
60% of loss of free-laccases after heating the
samples for 3 days. Laccase stabilization could
be of interest to future textile or cosmetic appli-
cations because of their potential for environ-
mentally friendly oxidation tech nologies.
Keywords: MLV Liposome; E n zymes; Laccases;
Encapsulation; Stability
1. INTRODUCTION
Enzymes have been used in different industries inclu-
ding textiles for washing, scouring, dyeing, etc. Protein
enzymes can interact with all products used in the process,
and their large 3D structure enables their interaction with
different chemical products in solution due to the variety
of side chains of the amino acids. Enzyme stabilization
has assumed considerable importance owing to the in-
creasing number of enzyme applications and to the need
for realizing their full potential as catalysts 1.
Liposomes are defined as a structure composed of
lipid vesicle bilayers enclosing an aqueous volume.
These structures have long been used as carrier systems
for the delivery of vaccines, therapeutic drugs and hor-
mones because of easy preparation, good biocompatibility,
low toxicity and commercial availability 2,3. Efficient
functioning of enzymes inside liposomes opens up new
possibilities of applications in biocatalysis and bioanaly-
tical tools 4-6. It has been observed that enzymes are
considerably stabilized within the nano-environment of
liposomes since they are protected from unfolding and
proteolysis. Liposomes can effectively protect enzymes
from aggression of external agents such as proteases 7.
In addition, enzymes entrapped in liposomes are stabi-
lized against unfolding forces owing to hydrophobic
interactions between the enzyme and th e liposome mem-
brane 8. Moreover, enzymes encapsulated inside lipo-
somes retain their activity even at very low concentra-
tions 9. Since liposomes are optically translucent, they
can be used as optical sensor elements 10. Novel lip-
osome-based nano-sized biosensor systems have been
prepared by encapsulating an enzyme using porin em-
bedded in the lipid membrane. As a result, the enzyme
activity within the liposome can be monitored using
pyranine as a fluorescent pH indicator 11 . Furthermore,
some enzymes such us horseradish peroxidase encapsu-
lated in liposomes have been directly detected without
lysis using Luminol chemiluminiscence 12.
There are two main areas of application of enzyme-
containing lipid vesicles: biomedicine (enzyme-replace-
ment therapy) and the food industry (cheese ripening
process). In both cases the lipid vesicles are carriers that
protect the enzymes from contact with blood or milk,
respectively 6. The stability effect of enzyme encapsu-

M. Martí et al. / Journal of Biophysical Chemistry 3 (2012) 81-87
82
lation in liposomes has been studied for many enzymes
in these two fields 6. However, few studies have been
performed with laccases that are used in textile and
cosmetic industries 13-15.
Laccases are of special interest because of their ability
to oxidise both phenolic and non-phenolic lignin related
compounds as well as other environmental pollutants,
which makes them very useful for biotechnologies. The
use of laccases in textiles is currently enjoying rapid
growth; they are used for decolorizing textile effluents
16,17, bleaching 18, dyeing 19, synthesizing dyes
20 and for modifying the surface of fabrics 21,22. In
the cosmetic field, laccases can replace H2O2 as an
oxidizing agent in dye formulation 23. Enzymes are
globular proteins whose catalytic function is due to their
three-dimensional confo rmation. For this reason, stability
strategies make use of compounds that avoid dismantling
or distorting protein 3D structures. Therefore, microen-
capsulation, particularly with liposomes, can be envi-
saged as a good strategy for stabilizing laccases. Of the
different existing oxidant enzymes, laccases have been
the subject of intensive research in the last decades due
to their low substrate specificity in the textile and
cosmetic fields as stated above. Therefore, new strategies
are needed for stabilizing and maintaining their enzyma-
tic activity.
Liposomes have also aroused a great deal of interest in
the textile industry 24,25 and in the cosmetic industry
26,27. This work seeks to shed light on the behaviour
of laccases microencapsulated in liposomes. Enzyme
stabilization was determined by the evaluation of the
thermostability of the free and entrapped laccases. Effi-
cient functioning of laccases inside liposomes would
open up new avenues for textile or cosmetic applications.
2. MATERIALS AND METHODS
2.1. Liposomes Formation and Enzime
Encapsulation
Liposome suspension of 2 wt% of phosphatidylcholine
(PC) and 10 wt% laccases solution (weight ratio of lipid
to laccase 1/5 lipid/laccases (LpLc)) and also another
suspension of 10 wt% of PC and 5 wt% of laccases solu-
tion (weight ratio of lipid to laccase 1/0.5 lipid/laccases
(LpLc)) were prepared using the film hydration method.
To obtain these liposome suspensions, 0.1g or 0.5g, re-
spectively of Lipoid S-100 (Lipoid GmbH, Germany)
were prepared in solution in 30 ml of chloroform. The
chloroform was then removed by a rotary evaporator
under reduced pressure. A thin film of lipid was observed
after the solvent were removed. The lipid film was hy-
drated with 5 ml buffer solution (pH 5) containing the
laccase solution, 5.86 mg/ml, commercial Trametes vil-
losa of Novozymes Spain S.A., (0.5 ml or 0.25 ml, re-
spectively) and, after 10 minutes sonication in a water
bath multilamellar vesicles (MLV) were obtained. Lipo-
somes suspensions of 2 wt% and 10 wt% of PC (Lp)
without enzymes were also prepared following the same
methodology but using only a buffer solution without
laccases.
The amount of protein was determined for the laccase
(Lc) solution and also for the lipid alone to ev aluate pos-
sible interferences. To quantify the laccases entrapped in
liposomes, LpLc was precipitated and separated from the
supernatant by centrifugation at 14000 RPM for 15 min-
utes using a Centrifuge 5415-Eppendorf (Germany). Af-
ter separation, the supernatant was retained. The precipi-
tate was filled at 1ml with buffer and agitated vigorously
and centrifuged again. This process was repeated two
more times and all supernatants were kept together (Sn
LpLc). Finally, the eppendorf with the precipitate lipo-
somes (V LpLc), the supernatant (Sn LpLc) and the full
LpLc solution, were filled with a 0.125% Triton X-100
solution, (octylphenol ethoxylated with 10 units of eth-
ylene oxide and active matter of 100%) supplied by
Tenneco S.A. (Spain), and agitated vigorously to solubi-
lise the lipid bilayer. The amount of protein was deter-
mined in all samples following the Bradford method in
order to obtain the amount of encapsulated laccase. The
formulations evaluated are summarized in Figure 1.
The Bradford method was used to quantify the protein
in these samples. It is based on the formation of a com-
plex between the dye, Brilliant Blue G, Sigma (USA),
and the proteins in solution, which produces an increase
Figure 1. Diagram of the protein quantification and of stability assays.

M. Martí et al. / Journal of Biophysical Chemistry 3 (2012) 81-87 83
in absorption at 595 nm and is proportional to the protein
present 28,29. To calculate the amount of protein, BSA
(Bovine Serum Albumin) from Sigma (USA) was used
as a standard.
2.2. Enzime Activity and Stability Assay
Enzyme activity (U) is defined as µmol of substrate
oxidized per min. Activity assay was performed using
ABTS. The assay mixture contains 0.0005M 2,2’-azino-
bis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) provided
by Sigma (USA), 0.1M sodium acetate buffer pH 5, and
a suitable amount of enzyme or liposome-enzyme. Lac-
case activity was assayed spectrophotometrically by
measuring the increase in absorbance at 420 nm ( 
420 =
3.6 × 104 M–1·cm–1) owing to the oxidation of ABTS 30.
When laccases were encapsulated in liposomes, the li-
posomes were solubilised with 10% Triton X-100. In all
cases 0.1 mL of sample which has lipids (LpLc, Sn LpLc
and V LpLc) was solubilised with 0.3 mL of 10% Triton
X-100 before the spectrophotometric assay. The stability
assessment was made by performing the activity assay
for three days following the graph in Figure 1. To in-
crease the experimental thermal conditions, the different
samples were heated at 60˚C for 120 minutes between
each activity assay. The results were obtained from trip-
licate assays.
3. RESULTS AND DISCUSSION
3.1. Liposome Formation and Enzime
Encapsulation
There are a number of methods that can be used for
the preparation of enzyme-containing lipid vesicles (li-
posomes) that are lipid dispersions that contain water-
soluble enzymes in the trapped aqueous space. A review
of these studies indicates that the most widely used vesi-
cle-forming amphiphiles are based on phosphatidylcho-
line 6. Moreover, encapsulation of enzymes in lipid
vesicles has been performed by using a variety of differ-
ent vesicle preparation methods 6. The dispersion of a
dry lipid film in an enzyme-containing aqueous solution
leads to the formation of a relative polydisperse vesicle
suspension with mainly large, multilamellar vesicles
(MLV). The preparation is not very reproducible because
the resulting size distribution and lamellarity very much
depends on the quality of the lipid film and on the way
this film is dispersed. Since most of these vesicles are
multilamellar, water-soluble enzymes can be localized
not only in the cen tral core but also in the aqueous inter-
lamellar spaces, resulting in relatively high encapsulation
efficiency. A number of studies on enzyme-containing
MLV have been carried out, e.g. with many enzymes, but
to our knowledge, not with Laccases. Therefore, lipo-
somes of 2% of phosphatidylcholine (PC) and 10% lac-
cases (1/5 lipid/laccases (LpLc)) and also 10% of PC and
5% of laccases (1/0.5 lipid/laccases (LpLc)) were pre-
pared using the dry lipid film hydration method de-
scribed in the experimental part. Multilamellar vesicles
were obtained.
The Bradford method was used to determine the
amount of enzymes in the vesicles and in the supernatant
solution of the original liposomes with laccases (LpLc)
for use in the enzyme activity assay. Accordingly, the
following formulations were prepared: the two liposomes
with laccases (LpLc), (1/5 and 1/0.5 lipid laccases) con-
taining 10% and 5% of laccase solution, respectively; the
two laccases only in buffer solution (Lc) containing 10
and 5% of laccase, respectively; and the two liposomes
in buffer solution, without enzymes, (Lp) with 2 and
10% PC in order to determine possible interferences of
the lipid compound. LpLc was centrifuged to separate
the precipitated vesicles from the supernatant. The pro-
tein content of all the solutions was evaluated. Signifi-
cant interferences were found between the phosphati-
dylcholine of the LpLc solutions and the Bradford re-
agent. This interaction was more marked with the latter
formulation (LpLc 1/0.5, with 10% of PC) because of the
high proportion of lipids with the result that the findings
were less reliable.
The results for the formulation with 2% of lipid and
10% of laccases LpLc (1/5) showed that the enzyme en-
capsulated accounted for 8%. When the proportion of
lipid increases LpLc (1/0.5) the enzyme encapsulation
reaches almost 15% (Table 1).
Enzyme entrapment is in general directly proportional
to lipid concentration 6,31,32. In our case, even only
Table 1. Laccase formulations, enzyme encapsulation, enzy-
matic activity (U/mL) and enzymatic activity percentages after
a thermal process.
Formulation 1/5 LpLc 1/0.5 LpLc
% Compounds Pc 2%, Lc 10% Pc 10%, Lc 5%
Encapsulation 8% 15%
U/ml % U/ml %
1st 50.0 100 25.0 100
2nd 35.1 70.2 19.2 76.8
Lc
3rd 20.5 41.0 14.4 57.6
1st 44.18 100 22.2 100
2nd 36.654 82.99 18.8 84.4 LpLc
3rd 24.471 55.38 17.2 77.5
1st 23.64 100 12.155100
2nd 22.271 94.21 10.43 84 Sn LpLc
3rd 19.722 83.43 8.271 67.8
1st 5.113 100 6.648 100
2nd 5.36 100 6.781 100
Enzymatic
Activity
V LpLc
3rd 4.98 97.4 5.95 89.5

M. Martí et al. / Journal of Biophysical Chemistry 3 (2012) 81-87
84
two lipid/laccases ratios were studied, but a clear in-
crease on encapsulation percentage with the increase on
lipid concentration was obtained. The encapsulation effi-
ciency of protein has been reported to depend on interac-
tion between the protein and the lipid bilayer 32. The
enzyme entrapment can be inc re ased b y ma nip ul at ion of
the liposomal lipid composition or by increasing the
lipid concentration, in order to favour electrostatic in-
teractions 32. Sometimes the entrapment efficiency
decreases with increasing the lipid content 31. The ad-
dition of more lipid increases the lamelarity of the vesi-
cle population rather than producing more vesicles of the
same lamelarity 31. Therefore, modification of lipid
composition and lipid/enzyme ratios would be the base
of further work with the aim to improve encapsulation
efficiency and to deep inside the protein-lipid interac-
tions.
Similar results have been reported for enzyme entrap-
ment efficiency of other enzymes in MLV. These varied
in most cases from below 5% 33-35 to about 15% 36.
Thus, although a smaller amount of enzymes and a lower
activity could be found in the liposome formulation of
10% of lipid and 5% of laccases LpLc (1/0.5), the higher
encapsulation obtained could demonstrate more clearly
the effect of the liposome on the stability of the encapsu-
lated laccases.
3.2. Enzime Activity and Stability Assay
Laccase activity was evaluated by the ABTS method
as detailed in the experimental part. The same amount of
laccases in liposomes and laccases in the buffer solution
of 5% and 10% were assessed. As expected, the enzyme
activity of the solution containing 10% of laccases (50.0
U/ml, 10% Lc) is approximately double that of the solu-
tion containing 5% laccases (24.9 U/ml, 5% Lc) since the
enzyme concentration is also the double. In both cases,
the presence of liposomes leads to a decrease in the en-
zyme activity of about 11.6% for 1/5 LpLc (44.2 U/ml,
10% LpLc) and 11.2 % for 1/0.5 LpLc (22.2 U/ml, 5%
LpLc). These decreases in laccase activities when for-
mulated with PC liposomes were expected because the
barrier of the lipid membrane diminished the activity of
the enzyme entrapped in the liposomes 37. It should be
borne in mind that formulations with a high amount of
enzymes are more prone to lose activity in the thermal or
proteolytical processes. Therefore, the similar percentage
of lost activity found for the two formulations could be
due to the high PC content in the 1/0.5 LpLc (10%Pc)
and to the high amount of Laccases in 1/5 LpLc formula-
tion (10% Laccases).
Even though en zyme activity is decreased in the initial
LpLc formulation when lipids were present, the stabili-
ties of the two formulations were assayed. The main aim
of this work was to study a possible increase in enzyme
stability in its formulation entrapped in liposomes. The
influence of enzyme entrapment on enzyme stability was
also investigated. To assess stability, a protocol was de-
signed to compare laccase stability in buffer solution (Lc)
and laccase stability formulated with liposomes (LpLc)
(Figure 1). In addition to evalu atin g th e to tal formulation
of laccases in liposomes, aliquots of the formulation
were centrifuged to separate the supernatant (Sn LpLc)
from the pellet (V LpLc) (in which laccases are encapsu-
lated) and the activities of the two samples were also
determined. To toughen the conditions, samples were
heated for 2 hours at 60˚C and the activity was measured
the following day. This process was continued for three
successive days.
The enzyme activity graph of the different samples ob-
tained during the three days of the experiment is shown
in Figure 2. The first day of the experiment during which
the samples were not heated, the laccases in the liposome
solution presented a lower activity than the laccases in
buffer solution, as discussed above. However, after 3
days, the LpLc formulation maintained higher activity
values than the free laccases Lc. The graph clearly shows
the stabilization obtained when laccases are bound to
liposome vesicles. Relative activity of other enzyme-
containing vesicles has also been demonstrated to retain
activity much longer than native enzyme 31 as in our
case. The stabilization of the supernatant should also to
be noted. However, higher stabilization was obtained for
the enzyme entrapped in the vesicles. The two formula-
tions show similar activity (5 - 7 U/ml) for the Laccases
entrapped in the vesicles. This occurs because the en-
zyme entrapment is double in this formulation despite
the presence of only 50% of the Laccase concentration in
the 1/0.5 LpLc.
The influence of the liposomes on the stability of lac-
case activity is clearly demonstrated by the percentage of
laccase activity of each sample during the whole stability
process (Table 1). The greater stabilization of the lac-
cases that are exclusively entrapped in the vesicles
should be noted. The activity loss of laccases entrapped
in the vesicles in the total stability process is lower than
10% when compared with 40% to 60% of activity of
free-laccases after heating the samples for 3 days. These
results confirm that the increase in the stability under-
gone by laccases in the liposome solution is mainly due
to the encapsulation degree of the enzymes.
In general, encapsulation of enzymes in liposomes has
demonstrated the enhancement of their stability versus
denaturizing 6,9. For example, other enzymes such as
amylogucosidase entrapped in multilamellar vesicles
(MLVs) composed of other phospholipids such as di-
palmitoylphosphatidylcholine (PPC) have been much
ore stable than free enzymes 38. The rate of hydroly- m

M. Martí et al. / Journal of Biophysical Chemistry 3 (2012) 81-87
85
Figure 2. Enzyme activity (U/ml) and their error bars (standard deviation) of lac-
cases in buffer solution (Lc), laccases in liposome formulation (LpLc), supernatant
of LpLc (Sn LpLc) and vesicles of LpLc (V LpLc), of 1/5 LpLc and 1/0.5 LpLc
during the stability a ssay.
sis is relatively low because of the low permeability of
substrate across the liposome bilayer 31. Moreover,
glucose oxidase encapsulated with phosphatidylcholine
and cholesterol liposomes has shown that the thermal
and proteolytic stabilities are also enhanced by encapsu-
lation in liposomes 38. Some studies indicate that the
interaction of the enzymes with the vesicle membrane is
based on the lipid vesicle assistance the refolding of un-
folded enzymes 319 of (6).
However, the activity of enzyme entrapp ed in the lipo-
some is markedly reduced by the permeability barrier of
the lipid membrane, resulting in a lower internal concen-
tration of the substrate. In order to overcome this prob-
lem, some authors have investigated th e permeabilization
of the wall of the liposome 9.
Currently there are generally two approaches to in-
crease the substrate permeability. One is to reconstitute
membrane channel proteins in the liposome bilayers
while the other is to utilize lipid/detergent hybrid mem-
branes (27 of 31). Exploiting the protective ability of
lipid nanocontainers in combination with controlled per-
meability by modified channels, could open new future
applications.
In our work, we present the stabilization behaviour of
another enzyme, the laccase whose use in textile and in
cosmetics, as stated in the Introduction, is growing rap-
idly 18,20,22,23,39. Stabilization and its relation with
the encapsulation degree are demonstrated. Development
of an effective system for laccase encapsulation has
lately deserved great attention to retain activity 40-43.
However, none of those works used vesicles formed with
phospholipids to encapsulate laccases. Further studies
will focus on modifying the lipid composition and/or
lipid/enzyme proportion to increase enzyme encapsula-
tion, diminish enzyme permeation with the aim of en-
hancing enzyme stabilization.
4. CONCLUSIONS
Laccases were microencapsulated in phosphatidylcho-
line MLV liposomes in different lipid/enzyme propor-
tions. Their encapsulation efficiency was evaluated, in-
dicating 8% of encapsulation when 1/5 lipid/laccases
were assayed versus 15% of encapsulation with 1/0.5
lipid/laccases. This demonstrates the increase in encap-
sulation efficiency at higher lipid/laccases ratios.
Enzyme activity showed a decrease of about 11% for
the two formulations, 1/5 lipid/laccases and 1/0.5 lipid/
laccases with respect to the laccases in buffer solution.
This decrease in laccase activity when formulated with
PC liposomes was expected given the barrier effect of
the lipid bilayers of the vesicles.
However, evaluation of the activity loss of laccases
entrapped in the vesicles during the thermal process
demonstrated an increase in stability undergone by lac-
cases in the liposome solution. This increase is much
more marked in the case of the laccases encapsulated in
the vesicles free of supernatant, which confirms the ef-
fect of the liposome encapsulation on the continued ac-
tivity of the enzyme. The use of phospholipidic lipo-

M. Martí et al. / Journal of Biophysical Chemistry 3 (2012) 81-87
86
somes in the laccases encapsulation warrants further
studies that modify the lipid composition and/or lipid/
enzyme proportion to increase enzyme encapsulation and
consequently enzyme stabilization. The study of other
lipid/laccases ratios would allow us to determine their
possible linearity with encapsulation efficiency and with
enzyme activity as it happen with other enzymes and
phospholipids [31,32]. Laccase stabilization could be of
considerable interest to future textile or cosmetic appli-
cations owing to their potential for environmentally
friendly oxidation technologies.
5. ACKNOWLEDGEMENTS
The authors are indebted to Ms. I. Yuste for technical support. The
authors also wish to thank G. von Knorring for improving the final
version of the manuscript. This work was supported by FCT (SFRH/
BPD/37045/2007; PTDC/CTM/100627/2008), QREN, COMPETE—
Programa Operacional Factores de Co mpetitividade na sua componente
FEDER.
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