Wood-rotting fungi, characterized by their ability to
degrade lignin and cellulose, are predominant microorganisms of wood degradation.
Some wood-rotting fungi, especially white-rot fungi, appear to have some
potential for bioremediation applications due to their non-specific system
which they have developed for depolymerization and mineralization of the
complex and recalcitrant polymer of lignin (Hatakka, 1994). As the lignin
degradation system of these fungi is not very substrate specific, they
are able to transform and sometimes completely mineralize a variety of
persistent environmental pollutants (Cameron et al., 2000). Among
such compounds there are many synthetic dyes characterized by high stability
in light and during washing, which give them recalcitrance to biodegradation
(Gramss et al., 1999). Between 10 and 15% of the total dyes consumed
in dyeing processes may be found in wastewater. Most of these compounds
are highly resistant to microbial attack. Therefore, it is hard to remove
them from effluents by means of conventional biological wastewater treatments.
The degradation of dyestuffs by fungi has been carried
out with either whole cultures or crude enzyme preparations of extracellular
ligninolytic enzymes. Decolourisation of azo, anthraquinonic, heterocyclic,
triphenylmethane and polymeric dyes and their partial mineralization by
enzymatic and non-enzymatic systems of these fungi have been reported
(Ferreira et al., 2000; Swamy and Ramsay, 1999). Several numbers
of white-rot fungi have been reported to produce the lignin-degrading
enzymes laccase, Lignin Peroxidases (LiP) and Manganese Peroxidases (MnP),
or at least one of these enzymes (Eggert et al., 1996).
Laccases (benzenediol: oxygen oxidoreductase, EC 220.127.116.11)
have very broad substrate specificity and they catalyze the removal of
a hydrogen atom from the hydroxyl group of ortho and para-substituted
mono and polyphenolic substrates and from aromatic amines by one-electron
abstraction to form free radicals capable of undergoing further reactions
such as depolymerization, repolymerization, demethylation, or quinone
formation (Thurston, 1994). The rather broad substrate specificity of
laccases may be additionally expanded by addition of redox mediators,
such as ABTS, 1-hydroxybenzotriazole (HBT) Claus et al.,
2002). Laccases from several fungi have been reported that can be used
for the treatment of effluents containing synthetic recalcitrant dyes.
T. hirsute and a purified laccase were able to degrade triarylmethane,
indigoid, azo and anthraquinone dyes used in dyeing textiles (Abadulla
et al., 2000) as well as 23 industrial dyes (odriguez et al.,
Pleurotus sajor-caju is one of popular commercial
mushrooms grown in several parts of Thailand and other parts worldwide.
Four laccase isozyme genes, Psc lac1, 2, 3 and 4 have been cloned
from this mushroom (Soden and Dobson, 2001). Apart from laccases, the
mushroom excreted ligninolytic enzymes MnP (Murugesan et al., 2006).
The Psc lac4 gene from P. sajor-caju has been cloned and
expressed in the heterologous host Pichia pastoris, purified and
biochemical characterized (Soden et al., 2002). Moreover, an extracellular
laccase was isolated and purified from this fungus grown in submerged
culture in a bioreactor and used to investigate its ability to decolorize
three azo dyes (Murugesan et al., 2006).
In the North Eastern area of Thailand, one of small popular
enterprises is silk. Many synthetic dyes have been used in silk dyeing
even though natural ones is considerate to alternatively use nowadays;
of course some are discarded into the environment. Therefore, in environmental
concerns it would be very valuable as if we could develop an easy and
uncomplicated way for dye-containing wastewater treatment. Due to availability
of this edible mushroom, it is made us interested to evaluate the potential
of dye decolourisation by its enzymes. In this present work, we have studied
the ligninolytic enzyme pattern, some characteristics of the crude enzyme
from P. sajor-caju and its potential to decolorize two structurally
MATERIALS AND METHODS
Microorganism: The white-rot fungus P. sajor-caju was obtained
from the commercial mushroom farm in Ka La Sin province (Northeast, Thailand).
The stock cultures were maintained on potato dextrose agar medium and
stored at 4°C with periodic subculture. All experiments were performed
at the Protein and Enzyme Technology Research Unit, Faculty
of Science, Mahasarakham University during August 2006 and February 2007.
Enzyme extraction, activity assays and characterization: The fungus
P. sajor-caju was grown on sorghum seed media without any mineral
supplementation for about 2 weeks at room temperature. The crude enzyme
was extracted with 50 mM sodium acetate buffer (pH 4.5), filtered through
sheet cloth to remove solid stuffs and then subjected to a centrifugation
at 3,000 rpm for 15 min to discard the precipitate. The clear supernatant,
crude enzyme was concentrated by ammonium sulfate precipitation at 0-80%
saturation. After centrifugation at 10,000 rpm, 15 min at 4°C, the
precipitate was re-suspended in 50 mM sodium acetate buffer (pH 4.5) prior
to enzyme activity assays and protein determination.
Laccase (EC 18.104.22.168) activity was spectrophotometric
assayed at 32°C of oxidized ABTS (blue green color cation radical)
as previously described (Khammuang and Sarnthima, 2007). Briefly, the
assay mixture contained 0.1 mM ABTS and 0.1 M sodium acetate buffer (pH
4.5). Oxidation of ABTS was followed by absorbance increase at 420 nm
(ε = 36,000 M-1 cm-1).
Lignin peroxidase (EC 22.214.171.124, LiP) activity was measured
according to Collins et al. (1996) using veratryl alcohol as substrate
in the presence of H2O2.
Manganese peroxidase (EC 126.96.36.199, MnP) activity was
measured the oxidation of MBTH (3-methyl-2-benzothiazolinone hydrazone)
and DMAB (3, 3-dimethylaminobenzoic acid) in the presence of H2O2
, a modified method of Ngo and Lenhoff (1980). The results were
corrected by activities in the reactions without manganese (Mn-independent
peroxidase, MnIP) where manganese sulfate was substituted by ethylenediaminetetraacetate
(EDTA) to chelate Mn present in the samples. All assays were performed
in duplicate, with an average sample mean deviation of less than 10%.
One unit (U) of enzyme activity was defined as the amount of enzyme that
catalyzed the formation of 1.0 μmol of product per minute.
Estimation of the laccase optimum pH was measured with
ABTS substrate in 50 mM sodium citrate-phosphate buffer (pH 2.0-8.0).
The optimum temperature was determined in the range of 30-60°C. The
pH stability was tested in the ranges of pH 3.0-8.0 while temperature
stability was performed at 30.0-60.0°C based on ABTS assay.
Protein assays: Protein concentration was determined by the Bio-Rad
Protein Assay Reagent (Bio-Rad) with bovine serum albumin as a standard.
Decolourisation experiments: Two dyes were tested for decolourisation
at a wavelength with maximum absorbance of each dye (610 nm for Indigo
Carmine and 524 nm for Methyl Red). Indigo Carmine concentrations were
used at 50-500 μM while Methyl Red concentration used from 5,000-50,000
μM. The enzyme activity in the reaction mixture was 1 U per 5.0x10-8
mole of dye. Experiments were monitored immediately using a JENWAY 6400
Spectrophotometer (LABQUIB, England) after enzyme addition and periodically
time interval. Dye decolourisation was expressed in terms of percentage
calculated according to the following equation.
An absorbance at λmax of
each dye immediately measured after enzyme addition.
An absorbance at λmax of
each dye after each time intervals.
Enzyme extraction and characterization: P. sajor-caju grown
on sorghum seed media with no mineral supplementation excreted extracellular
ligninolytic enzymes mainly laccase (3.340 U mL-1), some of
Mn-independent peroxidase, MnIP (0.217 U mL-1) and very small
amount of LiP (0.030 U mL-1). The laccase activity from this
fungus showed at least two isoenzymes with difference in electric mobility
as a result from a native- PAGE (Fig. 1A). The crude
enzyme when using ABTS as substrate, had a sharp pH optimum at 6.0 (Fig.
2A) and the optimal temperature at 40°C (Fig. 2B).
At temperature of 60°C, the laccase activity remained approximately
60% compared to those of the optimum temperature.
The enzyme stability experiments as shown in Fig.
3A revealed that storage pH had the enormous effect on stability of
laccase. It was found that the enzyme kept at 4°C in buffer pH 5.0
retained maximum activity. Within 180 min, the remaining activity was
more than 60% from the original. The laccase activity within 180 min was
remained less than 10% when kept in the buffer pH 3.0. Moreover, the activity
observed less than 20 and 40% when buffer pH 4.0 and 6.0 were used to
store, respectively. Neutral (pH 7.0) or basic condition (pH 8.0), the
results showed that more than half of activity was lost.
Temperature stability of the crude enzyme was also investigated
at the optimum pH condition. Among various temperatures studied, the best
condition for enzyme
PAGE analysis of the crude enzyme from Pleurotus sajor-caju stained
with ABTS substrate (A) and protein bands stained with coomassie
brilliant blue R-250 (B)
stability was at 30°C (Fig. 3B) and at this temperature
enzyme activity remained more than 95% after 120 min of incubation.
When kept the crude enzyme at 40°C, the laccase activity left above
80%, whereas only about 30% and less than 10% activity remaining when
the crude enzyme were kept at 50 and 60°C, respectively.
Effect of pH on dye decolourisation: Each dyes might be susceptible
to be efficiently degraded in different pH values, therefore two model
dyes with different chemical structures were evaluated for the pH dependent
decolourisation by the crude enzyme from P. sajor-caju. The results
showed that the optimum pH for Indigo Carmine and Methyl Red decolourisation
were shown in Table 1. Indigo Carmine dye could be decolorized
at pH 3.0-8.0 with a maximum decolourisation activity at pH 5.0, while
Methyl Red was decolorized at pH 4.0-8.0
pH optimum condition
(A) and temperature optimum (B) of the crude enzyme from Pleurotus
sajor-caju according to ABTS assay. Data were averaged from
a duplicate experiment
percentage of each dye at various pH valuesa
The reactions were performed using 50 mM sodium citrate-phosphate
buffer and dye 5.0x10-8 mole/Unit of enzyme at 32°C,
30 min. Data were averaged from a duplicate experiment
pH stability when
kept at 4°C (A) and temperature stability when kept at buffer
pH 5.0 (B) of the crude enzyme from Pleurotus sajor-caju.
Data were averaged from a duplicate experiment
with the highest percentage at pH 6.0. Therefore, pH
5.0 and pH 6.0 were chosen for following studies of Indigo Carmine and
Methyl Red decolourisations.
Effect of initial
Indigo Carmine concentration on decolourisation ability by the
crude enzyme from Pleurotus sajor-caju. The reactions were
performed in 50 mM sodium citrate-phosphate buffer, pH 5.0 and
1 Unit of enzyme at 32°C. Data were averaged from a duplicate
Effect of initial
Methyl Red concentration on decolourisation ability by the crude
enzyme from Pleurotus sajor-caju. The reactions were performed
in 50 mM sodium citrate-phosphate buffer, pH 6.0 and 1 Unit of
enzyme at 32°C. Data were averaged from a duplicate experiment
Effect of initial dye concentration on decolourisation Indigo
carmine: The indigoid dye Indigo Carmine decolourisation by crude
enzyme from P. sajor-caju at different initial concentrations (50-500
μM) as a function of time (Fig. 4). Within 180
min, at a fixed amount of enzyme it was observed that increasing initial
concentration the decolourisation ability was either slower or lower decolourisation
levels. At initial dye concentration of 50 μM, more than 80% of dye
decolourisation was achieved within 90 min and remained constantly after
that. The initial dye concentration that yielded the highest decolourisation
percentage was of 100 μM (>90% within 180 min). The initial concentration
of dye above this (250 and 500 μM) gave 60 and 10% decolourisation,
Methyl red: The decolourisation of Methyl Red, an azo dye (Fig.
5) which, unlike Indigo Carmine, decolourisation was very poor at
all initial dye concentration tested at optimum pH 6.0. The highest decolourisation
percentage, in the range of initial dye concentration tested, only 3.5%
was observed within 180 min.
White-rot fungus, P. sajor-caju from our experiment
grown on sorghum seed media without mineral supplementation showed that
there are at least two isoenzymes of laccases. The crude enzyme had optimal
activity with ABTS as substrate at 40°C and pH 6.0. These optimal
conditions are slightly different from the purified laccase investigated
by Murugesan et al. (2006), which reported that the optimal conditions
were 40°C and pH 5.0. However, the results from our work exhibited
the higher optimum temperature and less acidic pH than those of the purified
recombinant Psc lac4 which displayed optimal activity at 35°C
and pH 3.5 (Soden et al., 2002). According to these results, we
suggest that laccase in our crude enzyme may be the other isoenzymes of
P. sajor-caju different from previous reports.
Structurally different dyes were not decolorized to the
same extent. In present experiment, the Indigo Carmine, an indigoid dye,
was decolorized in a high percentage. At initial dye concentration lower
than 100 μM, more than 80% of dye decolourisation was achieved within
90 min. There has been a report that the decolourisation of this dye by
laccase from T. villosa was approximately 20% within 1 h (Basto
et al., 2007). This was relatively low when compared to the results
from present experiments. From the same report, they also observed that
the best results (more than 65% of decolourization) were attained when
the dye solution was treated with ultrasound and enzyme stabilized with
polyvinyl alcohol. It may point out that apart from the unique characteristics
of ligninolytic enzymes of different fungal species, laccase alone may
or may not be able to completely decolorize the Indigo Carmine solution.
This result was in agreement with other reported studies. T. hirsuta
immobilized on stainless steel sponge grown in a 1 L bioreactor supplemented
with 1 mM copper sulphate, the textile dye Indigo Carmine was almost totally
degraded in 3 days (Rodriguez et al., 2004).
The complete decolourisation of Indigo Carmine was only
achieved by a laccase from T. hirsuta when more than 30 h of treatment
with the enzyme immobilized in alginate beads (Dominguez et al.,
2005). Thus in our case, a good Indigo Carmine decolourisation might be
involved somehow with other enzymes apart from laccases. Mn-independent
peroxidase, another ligninolytic enzyme also found in the crude extract
of P. sajor-caju. Even though its activity was approximately 15
times less than laccase activity, it might concert in decolourisation
of Indigo Carmine dye somehow. However, the decolourisations of this dye
by the purified enzymes are still worth to study in order to more understand
On the other hand, Methyl Red, an azo dye, decolourisation
ability by the crude enzyme from P. sajor-caju was very low. A
low efficiency in decolourisation of some azo dyes, compared to other
dye types, was also reported for Thelephora sp. (Selvam et al.,
2003) which showed that a maximum of 19% Orange G was removed by laccase
(15 U mL-1) whereas LiP and MnP at the same concentration decolorized
13.5 and 10.8%, respectively. They also reported that a maximum decolourisation
of 12.0 and 15.0% for Congo Red and Amido Black 10B, respectively was
recorded by the laccase. However, decolourisation by the fungus in the
bioreactor provided higher efficiency of dye removal (Selvam et al.,
2003; Tavcar et al., 2006). Murugesan et al. (2006) reported
that the purified laccase from P. sajor-caju showed high efficiency
for decolorized azo dyes such as Acid Red 18 (90%), Acid Black 1 (87%)
and Direct Blue 71 (72%) within 24 h incubation with 10-12 U mL-1.
From present results, the crude enzyme from P. sajor-caju
showed fairly acidic pH of catalytic activity and fairly high temperature
optimum and quite stable at room temperature (30-40°C) as well as
its high potential in Indigo Carmine decolourisation, suggest that it
could be used for treatment of this type of industrial dye effluents.
In addition, other dyes in the same group or other groups of dyes such
as anthraquinonic and triphenylmethane dyes should also be further investigated.
However, redox mediators might be applied to improve decolourisation of
azo dye type. Purification and characterizations of each isoenzyme as
well as their dye decolourisation ability are underway in our laboratory.
This research was financially supported by a grant from
the Faculty of Science, Mahasarakham University (Fiscal year 2006).