Process Biochemistry 44 (2009) 435–439

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Recovery of laccase from the residual compost of Agaricus bisporus

in aqueous two-phase systems
Karla Mayolo-Deloisa a, Maria del Refugio Trejo-Hernández a, Marco Rito-Palomares b,*
a
Centro de Investigación en Biotecnologı´a, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos 62209, Mexico
b
Departamento de Biotecnologı´a e Ingenierı´a de Alimentos, Centro de Biotecnologı´a, Tecnológico de Monterrey, Campus Monterrey,
Ave. Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, Mexico

A R T I C L E I N F O A B S T R A C T

Article history: The potential use of aqueous two-phase systems (ATPS) to establish a viable protocol for the recovery of
Received 18 July 2008 laccase from the residual compost of Agaricus bisporus was evaluated. The evaluation of system
Received in revised form 8 December 2008 parameters such as poly (ethylene glycol) (PEG) molecular mass, concentration of PEG as well as salt and
Accepted 10 December 2008
system pH was carried out to determine under which conditions the laccase concentrates predominantly
to the top PEG-rich phase. PEG 1000–phosphate ATPS proved to be suitable for the primary recovery of
Keywords: laccase. An extraction ATPS stage comprising volume ratio equal to 1.0, PEG 1000 18.2% (w/w),
Laccase
phosphate 15.0% (w/w), system pH of 7.0 and loaded with 5% (w/w) of crude extract from residual
Residual compost
compost allowed the laccase recovery. The use of ATPS resulted in one-single primary recovery stage
Agaricus bisporus
Aqueous two-phase systems process that produced an overall yield of 95%. The results reported here demonstrated the potential
Protein recovery application of ATPS for the valorisation of residual material and the potential establishment of a
downstream process to obtain value added products with commercial application.
ß 2008 Elsevier Ltd. All rights reserved.

1. Introduction bioremediation, beverage (wine, fruit juice and beer) processing,

With the increasing concern to valorise the waste material
constantly generated, there is considerable interest in the
establishment of efficient processes to obtain valuable products
from residues. Currently, the processing of waste material
exploiting bioengineering strategies that will rapidly deliver
commercial products will definitively draw attention from
industry. The establishment of processes that permit the recovery
of commercially attractive products from a wide variety of residues
is essential. In this context, the potential recovery of enzymes
products from microbial residues represents a very interesting
case that has been addressed before and continues to raise interest.
The design of efficient recovery and purification protocols that
allow the recovery of enzymes from residues is one of the key goals
in the field. Particularly, in this research laccase produced by
Agaricus bisporus was selected as a representative model of this
group of value added products of commercial interest that can be
recovered from microbial residues. Laccases are multi-copper
oxidases that catalyze the one electron oxidation of several
aromatic substrates with the simultaneous reduction of dioxygen
to two molecules of water [1]. Laccases can be used in
* Corresponding author. Tel.: +52 81 8328 4132; fax: +52 81 8328 4136.
E-mail address: mrito@itesm.mx (M. Rito-Palomares).
ascorbic acid determination, sugar beet pectin gelation baking and
as biosensor [2]. Laccase activity has been extensively demon-
strated in more than 60 fungal strains like A. bisporus, Pleurotus
ostreatus and Trametes versicolor [3,4].
A. bisporus is considered one most important edible mushroom
from the economic perspective. It has been reported that during
the growth of the A. bisporus mycelium on composted wheat straw,
large amounts of laccases are produced [5–7]. After fruit body
harvesting, a considerable amount of residual compost is discarded
as by product. This residue is a potential source of ligninolytic
enzymes like laccase [6,7].
Several methods for purification of laccase have been reported
that consist mainly of ultrafiltration, dialysis, one or more types of
chromatography (i.e. exclusion, ion exchange) and electrophoresis
[5,8–14]. The use of multi-step process approach results in low
yield, high costs of supplies and operation. To overcome some of
the disadvantages attributed to the established protocols for the
recovery of laccase from residual compost, the use of aqueous two-
phase systems (ATPS) [15] is proposed as an attractive alternative
in this research work. ATPS, assembled from a mixture of polymers
(e.g. poly (ethylene glycol); PEG) and salts (e.g. phosphate or
sulphate), result in two-phases for the extraction of bio-molecules.
ATPS posses several advantages compared to the conventional
process, including bio-compatibility, ease of scale-up and low cost
[15,16]. In the present research, a practical approach, which

1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2008.12.010
436 K. Mayolo-Deloisa et al. / Process Biochemistry 44 (2009) 435–439

exploits the known effect of system parameters such as PEG and

salt (phosphate and sulphate) concentration and the molecular
mass of PEG upon product partition, was used. This approach was
followed to evaluate the feasibility of using ATPS for the recovery of
laccase from residual compost of A. bisporus as a first step in the
development of a prototype bio-process. The research was
conducted using a crude extract of the residual compost of A.
bisporus to obtain different conditions where the target product
and the contaminants partitioned preferentially to opposite
phases. These conditions were then used to evaluate the effect
of increasing crude extract concentration on the system perfor-
mance.

2. Experimental

2.1. Crude extracts from residual compost of A. bisporus

Fig. 1. Effect of pH on the enzyme activity of laccase recovered from the residual
Residual compost of A. bisporus was kindly provided by Dr. Hermilo Leal-Lara
compost of Agaricus bisporus.
from National Autonomous University of Mexico (UNAM). The residual compost
(2.0 g) was mixed with distilled water (3.0 mL). The resulting mixture was kept at
4 8C for 24 h. The solid phase was recovered and pressed manually to increase the
3. Results and discussion
amount of liquid phase obtained. Subsequently, the liquid phase was passed
through a mesh (0.8 mm) and centrifuged (10,000  g) as described by Trejo-
Hernandez et al. [7]. The resulting liquid was referred as crude extract from residual Initially, laccase rich extracts were subjected to different pH
compost of A. bisporus. The laccase enzymatic activity was estimated from the crude environments to estimate process extraction conditions exploiting
extract prior to lyophilization and storage at 20 8C until use. ATPS. Fig. 1 shows the impact of pH on the enzyme activity of
laccase recovered from the residual compost of A. bisporus. These
2.2. Effect of pH on enzyme activity
results can be utilized to define the pH of the ATPS for the potential
Prior to aqueous two-phase experiments the effect of pH on the enzyme activity extraction of laccase. The results of Fig. 1 show that the maximum
of laccase from crude extract of A. bisporus was evaluated. A mixture of lyophilized enzyme activity is obtained at pH range of 4.0–4.5. Decrease of pH
crude extract with distilled water (10%, w/w) was prepared. pH was changed from below 4.0 and an increase above 4.5 resulted in a reduction of
2.5 to 5.5 and 5.5 to 7.0 using sodium acetate–acetic acid buffer (0.1 M) and
laccase activity. The decrease in laccase activity seen for pH values
phosphate buffer (0.1 M) respectively. Samples were taken from each system pH for
further biochemical analysis. above and below 4.0–4.5 may be the result of either lower
solubility or loss of activity (or both). From these results it was
2.3. Aqueous two-phase experiments
decided that ATPS characterized by system pH below 7.0 will be
initially utilized for the potential extraction of laccase from the
For the aqueous two-phase experiments, systems were selected based upon residual compost of A. bisporus.
previous experiences [15,17] to give a volume ratio of 1.0 and a fixed weight of 5.0 g.
In order to evaluate the effect of system pH below 7.0 on the
The strategy behind the selection of the experimental systems is well described
elsewhere [15]. The system tie-line length (TLL), which represents the length of the recovery of the enzyme from the top PEG-rich phase, PEG–sulphate
line that connects the compositions of the top and bottom phases in a phase ATPS were utilized. Based upon previous experience establishing
diagram for a defined system, was calculated as described before [18]. initial process conditions [15], low molecular mass of PEG was
Predetermined quantities of stock solutions of potassium phosphate and poly
selected (i.e. 1000 and 1450 g/mol). PEG–salt systems characterized
(ethylene glycol) (PEG, Sigma Chemicals, St. Louis, MO, USA) of nominal molecular
masses 1000, 1450, 3350 and 8000 g/mol were mixed with lyophilized crude by molecular mass lower than 1000 g/mol promote the accumula-
extract from residual compost of A. bisporus (the amount of crude extract added to tion of cell and cell debris at the top PEG-rich phase [15]. Fig. 2 shows
the ATPS represent the 1.0–7.0% (w/w) of the total system) to give the desired PEG/ the effect of system pH on the recovery of laccase from the residual
salt composition with a final weight of 5.0 g. Additional, 5.0 g experiments were
conducted using PEG–sulphate systems to evaluate the effect of system pH upon
the behaviour of laccase from crude extract of A. bisporus. Adjustment of the pH
(from 3.0 to 7.0) was made by the addition of 1.0 M orthophosphoric acid or
potassium hydroxide if needed. Complete phase separation was achieved by low-
speed batch centrifugation at 1500  g for 10 min. Visual estimates of the volumes
of top and bottom phases were made in graduated tubes. The volumes of the phases
were then used to estimate the experimental volume ratio (Vr, defined as the ratio
between the volume of the top phase and the bottom phase). Samples were
carefully extracted from the phases (top and bottom phase) and analyzed. The top
and bottom phase recovery was estimated as the amount of the target product
present in the phase (volume of the phase  volumetric enzymatic activity in the
phase) and expressed relative to the initial total activity loaded into the system.
Purification factor was estimated as the ratio of the specific activity of laccase after
and before the extraction stage. The specific activity of laccase is expressed relative
to the total amount of proteins. Results reported are the average of three
independent experiments.

2.4. Analytical techniques

The enzymatic activity was estimated following the change in optical density at
436 nm using ABTS as a substrate and the extinction coefficient
(e436 = 29,300 M 1 cm 1) as described by Tinoco et al. [19]. Briefly, the assay
mixture consisted of 1.775 mL of acetate buffer (0.1 M, pH 4.0), 0.2 mL of substrate
(ABTS, 1 mM) and 0.025 mL of enzyme extract-sample. One unit of enzyme activity
was defined as the amount of enzyme required to oxide 1.0 mmol of substrate per
minute. Protein concentration from the phases was determined using the method of
Bradford [20].
Fig. 2. Effect of system pH on the recovery of laccase from the residual compost of A.
bisporus in PEG–sulphate ATPS. The selected systems comprising PEG 1000 18% (w/
w), sulphate 15% (w/w) (^) and PEG 1450 18.3% (w/w) and sulphate 11% (w/w) (~),
exhibited volume ratio (estimated from blank systems) and tie-line length (TLL)
equal to 1.0 and 40% (w/w) respectively. The concentration of crude extract from
the compost of A. bisporus in the ATPS was kept constant at 1.0% (w/w).
 K. Mayolo-Deloisa et al. / Process Biochemistry 44 (2009) 435–439 437

compost of A. bisporus in PEG 1000 and PEG 1450–sulphate ATPS. In
general, increasing the system pH caused an increase in the top
phase recovery of laccase from the residual compost. Despite the
known laccase stability at acid pH (3–5) [21] (see also Fig. 1), higher
enzyme recovery from the top phase was obtained when the pH
increased above 4.0. Several authors have discussed the influence of
system pH on protein partition behaviour [17]. In general, these
reports attributed the increase in the protein concentration in the
top phase and a decrease in the bottom phase with increasing pH to
free volume effects and potential changes in the structural integrity
of the proteins [15,17]. Laccase from A. bisporus is an acidic enzyme,
with an isoelectric point (pI) around pH 4.0 [21]. When the pH of the
extraction ATPS system is increased above of the pI, the laccase
surface charge becomes negative. As a result an increase in the
partition coefficient is observed. This behavior was observed with
other acidic enzymes [22–25]. Such partition behaviour indicates
that laccase is a hydrophobic protein. Andrews et al. [26] have
studied the protein behaviour in ATPS and concluded that the
hydrophobicity and charge of protein are important factors in the
enzyme partition.
Laccase exhibited a better partitioning at pH 7.0 under PEG-rich
environments. PEG–sulphate systems are better suited for pH
values below 6.5, while PEG–phosphate systems are more stable at
pH values above 7.0. Therefore, for further evaluation of ATPS
performance, at 7.0, PEG–phosphate systems are recommended
[15]. Furthermore, in an initial comparison, the enzyme recovery
from PEG–phosphate systems at pH 7.0 resulted superior from
those obtained from PEG–sulphate systems (see Fig. 2 and Table 1).
The enzyme recovery from PEG 1000 and PEG 1450–phosphate
systems was 80% and 72%, respectively. In contrast, in PEG 1000
and PEG 1450–sulphate systems the recovery of laccase was 74%
and 62%, respectively. Thus it was decided to continue the
characterization of the behaviour of laccase obtained from the
residual compost of A. bisporus, at pH 7.0, in ATPS formed by PEG
and phosphate. PEG–phosphate systems at pH 7.0 can be obtained
by the appropriate mixture of the phosphate–salt that form the
phases. Further evaluation of ATPS performance at pH above 7.0
was not pursued due to the potential application of the enzyme for
bioremediation purpose and to avoid the addition of preparative
stage to the process (e.g. adjustment of the system pH). PEG–
phosphate systems are the most used and characterized systems in
the literature [15]. Initially, the effect of increasing TLL upon the
partition behaviour of laccase was evaluated. It has been reported
[17] that changes in the TLL affect the free volume available for a
defined solute to accommodate in the phase and as a consequence
in the partition behaviour of such solute in the ATPS.
The effect of increasing TLL upon laccase obtained from the
residual compost of A. bisporus when PEG of four different
molecular mass (i.e. 1000, 1450, 3350 and 8000 g/mol) was used,
is illustrated in Table 1. For all these systems, volume ratio and
system pH were kept constant at 1.0 and 7.0, respectively. Overall
top phase recovery of laccase decreases with the increase in TLL. An
increase in the TLL of an ATPS caused the free volume in the bottom
phase to decrease [27] and as a result, the solutes in the lower
phase may be promoted to the partition to the top phase.
Consequently, the increase of contaminant proteins that concen-
trate in the top phase with increasing TLL is possible and as a result
the amount of the target product that can be accommodate in such
phase may be negatively affected [17]. Furthermore, an increase in
TLL decreases the volume available in the top phase for solute
dissolution. As a result the amount of soluble solute (target and
contaminants) in such phase decreases affecting the potential top
phase product recovery. It is also important to note that the top
phase recovery of laccase decreases when high PEG molar mass
were used (see Table 1). The effect of increasing molecular mass of
PEG upon protein partition behaviour has been explained based
upon the protein hydrophobicity [28] and phase excluded volume
[17]. In this case, the decrease in laccase top phase recovery when
high PEG molar mass were used may be explained by a migration of
the enzyme from the top phase to the bottom phase or interface.
ATPS with low molecular mass of PEG (i.e. PEG 1000 and PEG 1450)
show the best laccase top phase recovery. ATPS using PEG 1000
(TLL 40 and 45%, w/w) and PEG 1450 (TLL 36 and 39%, w/w) were
selected to evaluate the effect of the increase in the concentration
of the extract crude loaded to extraction system on the recovery of
laccase.
An increment in the concentration of crude extract fractionated
via ATPS may be of benefit to the potential intensification of the
proposed ATPS process. The effect of the concentration of crude
extract of the residual compost of A. bisporus upon the laccase
recovery is shown in Table 2. It is clear that ATPS was able to
fractionate concentrated crude extract. Furthermore, top phase
laccase recovery increased when the concentration of the crude
extract was raised. PEG of low molecular mass (for example, 1000
and 1450 g/mol) helps to overcome saturation problems at the top
phase [17,27,29]. For ATPS selected (i.e. PEG 1000 and PEG 1450),
the best performance was obtained at a concentration of crude
extract of 5% (w/w). Apparently, when higher concentration of
crude extract were used (i.e. 7%, w/w) a negative impact on the
laccase recovery was observed. This behaviour can be attributed to
the saturation of the top phase system due to the complexity of the
crude extract from the residual compost of A. bisporus. It is clear

Table 1
Effect of system tie-line lengths and molecular mass of PEG on the recovery of laccase from the residual compost of A. bisporus in PEG–phosphate ATPS.

System Molecular mass of PEG (g/mol) % PEG (w/w) % Phosphate (w/w) TLL (% w/w) Top phase recovery (%)

1 1000 18.2 15.0 40 80  1.3

2 18.9 16.0 45 78  2.8
3 22.2 19.0 55 67  1.2
4 24.1 20.1 59 49  1.5
5 1450 15.1 13.0 36 72  1.6
6 17.5 14.3 39 72  2.0
7 21.9 18.0 51 53  2.4
8 23.0 19.8 56 40  0.8
9 3350 18.0 15.0 43 59  0.7
10 21.9 18.0 56 44  3.0
11 22.2 19.0 58 40  1.0
12 24.1 20.1 62 39  7.0
13 8000 15.0 11.0 30 40  1.0
14 16.5 11.5 33 39  2.0
15 19.0 12.5 41 35  3.0
16 24.1 20.1 63 32  2.0

The system pH of the selected systems was kept constant at 7.0. The concentration of crude extract from the compost of A. bisporus in the ATPS was kept constant at 1.0%
(w/w).
438 K. Mayolo-Deloisa et al. / Process Biochemistry 44 (2009) 435–439

Table 2
Effect of crude extract concentration on the recovery of laccase from the residual compost of A. bisporus in ATPS.

Molecular mass of PEG (g/mol) % PEG (w/w) % Phosphate (w/w) TLL (% w/w) Crude extract concentration (% w/w) Top phase recovery (%)

1000 18.2 15.0 40 1.0 80  1.3

2.0 84  4.6
5.0 95  6.8
7.0 43  0.8
18.9 16.0 45 1.0 78  2.8
2.0 81  4.0
5.0 91  5.0
7.0 43  1.5

1450 15.1 13.0 36 1.0 72  1.6

2.0 94  3.0
5.0 80  2.2
7.0 53  1.5
17.5 14.3 39 1.0 72  2.0
2.0 64  5.0
5.0 78  3.8
7.0 46  2.2

Table 3
Direct comparison of the developed and the previously reported processes for the recovery of laccase from different sources.

Microorganism Main primary recovery stage Process yield (%) Purification factor Type of fermentation Reference

Agaricus bisporus ATPS 95 2.48 Solid (residual compost) This research

Agaricus bisporus Ultrafiltration 88 2.7 Liquid Wood [5]
Pycnoporus sanguineus Ultrafiltration 87 2.14 Liquid Lu et al. [8]
Agaricus blazei Ultrafiltration 83 1.6 Liquid Ullrich et al. [9]
Panus tigrinus Ultrafiltration 93 1.18 Liquid Quaratino et al. [10]
Volvariella volvacea Ultrafiltration 94 1.1 Liquid Chen et al. [11]
Trametes versicolor Ammonium sulphate precipitation 56 2.24 Liquid Han et al. [12]
Lentinula edodes Ammonium sulphate precipitation 88 1.98 Liquid Nagai et al. [13]
Perenniporia tephropora Ammonium sulphate precipitation 80 1.03 Liquid Ben Younes et al. [14]

Selected ATPS (TLL 40% (w/w), PEG 1000 18.2% (w/w), phosphate 15% (w/w), loaded with a concentration of crude extract of 5% (w/w)).

from these studies that when a concentration of crude extract of 5%
(w/w) was used, ATPS characterized by PEG of 1000 g/mol
molecular mass showed a superior performance compared to that
from the PEG 1450 systems. Selected extraction conditions (i.e.
Vr = 1.0, PEG 1000 18.2% (w/w), phosphate 15% (w/w), TLL of 40%
(w/w) and system pH of 7.0) resulted in a laccase top phase
recovery of 95%. A further direct comparison based upon the
purification factor and enzyme yield achieved between the
proposed protocol for the primary recovery of laccase from the
residual compost of A. bisporus and previously reported processes
(Table 3) highlights the advantages of the ATPS approach. The
purification factor obtained from our work is similar to that
reported by Wood [5] and higher than those obtained for laccases
produced by other fungi. The proposed process involves a one-
single extraction stage to fractionated complex residual compost.
The previously reported protocols involve the recovery of laccases
when liquid fermentation was used. The use of solid residual
compost for the recovery of laccase has been slightly studied due to
the complexity of mixture. To date, no previous studies known by
the authors have been reported that exploit the potential use of
ATPS for the recovery laccase from a complex waste material such
residual compost. It is clear that, for certain residual material, this
process opens the way to further processing such material as a first
step to potentially obtain value added products.
4. Conclusions
This study reports the processing of residual compost from A.
bisporus in aqueous two-phase systems for the potential primary
recovery of laccase. It has been shown that parameters such as tie-
line length, molecular weight of PEG, type of salt and system pH,
influence the recovery of laccase from the top PEG-rich phase. PEG
1000–phosphate ATPS proved to be suitable for the primary
recovery of laccase, since top phase recovery was higher than 90%.
The operating conditions selected for the ATPS resulted in a one-
single extraction process for the potential recovery of laccase from
residual compost of A. bisporus. Overall, the results reported here
demonstrated the potential application of ATPS for the primary
recovery of laccase from residual compost material as a first step
for the valorisation of such materials and the potential establish-
ment of a downstream process with commercial application.
Acknowledgements
The authors wish to acknowledge the financial support of
Tecnologico de Monterrey, Bioengineering and Nano-bioparticles
Research Chair (Grant CAT161). The authors are very grateful to the
assistance of the Hermilo Leal-Lara, Mayra Cisneros, Leobardo
Serrano-Carreón and Mario Caro.
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