White rot fungi are well recognized as lignin degrader. These fungi secrete
ligninolytic enzymes for hydrolyzing their substrates. Ligninolytic enzymes
mainly contain lignin peroxidase, manganese-dependent peroxidase and laccase.
Some strains of white rot fungi produce all three ligninolytic enzymes but some
produce only one or two enzyme (Baldrian, 2006). Lignin
peroxidase and manganese-dependent peroxidase require hydrogen peroxide as co-substrate
but there is no need for laccase. Therefore, laccase is more practically used
when it needs for applications. Laccase catalyzes the oxidation of several substrates
including phenolic compounds, aromatic amines, thiols and some inorganic compounds
using molecular oxygen as electron acceptor (Pezzella et
al., 2009). The low substrate specificity makes this enzyme interesting
for biotechnology purposes in various industries such as pulp and paper and
textiles and bioremediation of industrial pollutants (Arora
and Sharma, 2010). High amount ligninolytic enzymes productions in considerable
low cost substrates are of interest. In addition researches have been investigated
to find laccase with new properties or interesting properties for their applications.
The whiterot fungus, Lentinus polychrous is edible mushroom which is
popular to eat in Northeast and North of Thailand. In our laboratory, we have
reported ligninolytic enzymes activity of crude extract of the fungus grown
on Solid-state Fermentation (SSF) and their potential uses in dye decolorization
(Khammuang and Sarnthima, 2007; Sarnthima
et al., 2009). In this study, therefore, we extended investigated
production of ligninolytic enzymes from Lentinus polychrous on SSF in
the presence of copper(II)sulfate as laccase inducer as well as evaluated their
ability in back liquor decolorization.
MATERIALS AND METHODS
Production and preparation of crude enzyme: Lentinus polychrous
Lév. was kindly donated by Rujira mushroom farm, Ka La Sin province,
Thailand. It was maintained at 4°C on Potato Dextrose Agar (PDA) plates
and sub-cultured every 1-2 months. Ligninolytic enzymes were obtained by culturing
the fungus on solid substrates of rice husk and bran as previously reported
by Sarnthima et al. (2009). The laccase enzyme
was induced by adding copper two ions (CuSO4 0.04-2.4 μmol g-1
solid substrate in distilled water) into the culture. The fungal cultures were
performed at room temperature (28±2°C) for 5 weeks. The enzymes were
extracted from the fungal culture media using distilled water in the extraction
ratio of 1 g of the fungal culture media per 3 mL of distilled water. The extraction
was stirred by a stirring rod manually for 10 min. The filtered through a double
layer of cheesecloth was then centrifuged at 10,000 rpm for 20 min at 10°C
to get a clear supernatant called the crude enzyme.
Protein determination and ligninolytic enzymes assay: Protein content
in crude extract was determined by the method of Bradford
(1976) using Bio-Rad protein assay reagent and Bovine Serum Albumin (BSA)
as a protein standard.
Laccase (Lcc) activity was determined using 2,2-azinobis-(3-ethylbenzo-6-thiazoline-sulfonic
acid) (ABTS) as a substrate. Briefly, the reaction was performed in 0.1 M sodium
acetate buffer (pH 4.5) at 32°C for 10 min as previous described by Khammuang
and Sarnthima (2007). One unit of laccase activity is the amounts of enzymes
that oxidizes 1.0 μmole substrate and generate 1.0 μmole product per
minute at assay condition.
Lignin peroxidase (LiP) activity was measured by the oxidation of Veratyl Alcohol
(VA) in 0.1 M phosphate buffer, pH 6.5 in the presence of H2O2.
After incubation at 32°C for 10 min, the absorbance was read at 310 nm (ε
= 11.4x103 M-1 cm-1) or using the principle
of Azure B decolourization (Sarnthima et al., 2009).
Manganese peroxidase (MnP) activity was measured as described previously (Sarnthima
et al., 2009) using 3-methyl-2-benzithiazolinone hydrazone (MBTH)
and dimethylaminobenzoic acid (DMAB) as substrates in 0.1 M sodium acetate buffer,
pH 4.5 in the presence of H2O2 and Mn2+. The
absorbance was read at 590 nm (ε = 5.3x103 M-1 cm-1).
Manganese-independent Peroxidase (MIP) activity was measured using the same method as the manganese peroxidase activity but adding EDTA for cheated ion of Mn2+. The real MnP activity was corrected by subtraction of MIP activity from peroxidase activity.
Carbohydrate degrading enzymes assay: The xylanase activity was determined
by the method of Bailey et al. (1992). The substrate
solution, 1% birchwood xylan solubilized in 0.5 M citrate-phosphate buffer (pH
6.0). The reaction mixture consisted of 1.8 mL substrate solution and 0.2 mL
properly diluted enzyme. After 5 min of incubation at 50°C, the liberated
reducing sugars (xylose equivalent) were estimated by the dinitrosalicylic acid
method (DNS) according to Miller (1959). One unit of
xylanase is defined as the amount of enzyme releasing 1 μmole of reducing
sugar per minute per milliliter under the assay conditions described.
The cellulase activity was determined as Filter Paper Activity (FPA) according
to Ghose (1987). Whatman No. 1 filter paper strip (1.0x6.0
cm) was incubated with 0.5 mL of crude extract at appropriate dilution in 0.05
M Na-citrate buffer (pH 4.8) and with 1 mL sodium citrate buffer. After 60 min
of incubation at 50°C, the liberated reducing sugars (glucose equivalent)
were estimated by the DNS method according to Miller (1959).
Beta-glucosidase activity was determined by the hydrolysis reaction of p-nitrophenyl-β-D-glucoside
(Glcβ-O-pNP) in 0.1 M citric acid-0.2 M Na2HPO4 (McIlvaine
buffer), pH 5 at 30°C and measured the release of p-nitrophenol (p-NP) spectrophotometrically
at a wavelength of 400 nm (Srisomsap et al., 1996).
One unit of Beta-glucosidase activity is defined as the amount of enzyme that
releases 1 μmole of p-NP per minute.
Enzyme characteristics: Optimum pH and optimum temperature for catalysis of the crude enzyme were investigated using ABTS as laccase substrate. For pH optimum, the assay reactions were performed as previously described but in various pH conditions ranging from pH 2-9. For temperature optimum, the reactions were incubated at various temperatures ranging from 30-75°C.
The crude enzyme was studied pH stability by incubation in buffers different pH ranging from 3-8 including in distilled water at 32°C, aliquots were taken periodically for laccase activity assay. Temperature stability was done by incubating the crude enzyme in buffer pH 7.0 at various temperatures of 30, 40, 50 and 60°C, aliquots were withdrawn periodically for laccase activity assay.
Polyacrylamide Gel Electrophoresis (PAGE) was performed under nondenaturing
conditions. The separating and stacking gels contained 12 and 4% acrylamide,
respectively. The gel was run according to Ornstein (1964)
and Davis (1964) at constant volts of 100 V per gel.
The gels were stained to visualize ligninolytic enzymes activity by using ABTS
and syringaldazine for laccase activity and MBTH+DMAB+ H2O2+Mn2+
for peroxidase activity. The gel was also stained with coomassie brilliant blue
R-250 for protein distributions.
Black liquor decolorization: Biodegradation of black liquor by the crude
enzyme from L. polychrous was also investigated in order to confirm enzymatic
delignification. Degradation of by-products was measured by a spectrophotometric
technique. The solutions were scanned over the wavelength ranging from 200 to
600 nm by a UV-visible spectrophotometer (Lara et al.,
2003). Reaction mixtures (2 mL) contained 0.2 U mL-1 laccase
activities, the suitable diluted black liquors and without or with some mediators
in 0.1 M sodium acetate buffer (pH 4.5), were incubated at 32°C for 48 h.
The reactions were periodically monitored the decrease in absorbance at the
maximum absorption wavelength (λmax). For black liquors decolorization,
these were used to calculate decolorization percentage according to the following
equation as previously described by Khammuang and Sarnthima
Where, A0 is an absorbance at the maximum absorption wavelength of the black
liquor immediately measured after adding the enzyme solution and At is an absorbance
at the maximum absorption wavelength of the black liquor after each time intervals.
RESULTS AND DISCUSSION
Extracellular lignin degrading enzymes production: The fungus L.
polychrous was cultured on solid substrate of rice husk and bran (1:2 by
weight) in total about 45 g substrate containing 0, 0.04, 0.16, 0.8 and 2.4
μmole CuSO4 as laccase inducer. During cultured at room temperature
the ligninolytic enzymes were extracted by distilled water periodically for
5 weeks. Then, the extract was centrifuged and the supernatant was measured
for the total volume, protein contents, ligninolytic enzymes activities including
laccase, MnP, MIP and LiP. The results showed in Fig. 1a-d.
The crude enzymes from without and with inducer CuSO4 (0.04-2.4
μmole CuSO4/g solid substrate) showed similar in the protein
pattern (Fig. 1a) but clearly different in ligninolytic enzymes
production (Fig. 1b-d). However, the fungus
cultured in the presence of CuSO4 as inducer showed protein concentration
slightly higher than those of the absence one at all of observed times (Fig.
For laccase activity assay, the result showed a rapid increasing of laccase
activity in the week of 3-4 cultivations (21-28 days) and after that, it is
static or slightly decreasing (Fig. 1b).
|| Protein content (a) Laccase, (b) MIP, (c) and MnP activity
d) in the absence and presence of 0.04, 0.16, 0.8 and 2.4 μmole CuSO4
per gram solid substrate at different times of cultivation
It was found that in the presence of CuSO4 inducer, laccase activity
was increased to its maximum above 4 U mL-1 at CuSO4 concentration
ranging from 0.04-0.8 μmole per gram solid substrate with laccase specific
activity around 8 U mg-1. Culture of 2.4 μmole CuSO4
per gram solid substrate, laccase activity was lower. Therefore, L. polychrous
culture in this rice husk and bran media in the presence of CuSO4
inducer between 0.04-0.8 μmole per gram solid substrate for 28 days seemed
to be the most suitable condition for laccase production. At 35 days of cultivation,
laccase activity from all cultures with CuSO4 induced was decreased,
except culture of the fungus without inducer which activity was slightly increased
(Fig. 1b). Copper(II)sulfate is most known laccase inducer
of white rot fungi. This has been reported as a good inducer in many fungal
strains and being involved with isoenzymes production (Palmieri
et al., 2000; Levin et al., 2002;
Cordi et al., 2007).
For lignin peroxidase activity assay, the result showed no activity of this
enzyme in all of cultivation conditions when using VA or Azure B as substrates
(assayed only in 28 days of cultivation conditions). Since, this fungus may
not have gene of LiP enzyme or if any, it seemed not expressed. Some white rot
fungi produce only laccase, no MnP and LiP for example laccase producing Pycnoporus
cinnabarinus (Eggert et al., 1996; Geng
and Li, 2002), laccase and MnP producing Ganoderma lucidum (DSouza
et al., 1999).
For manganese independent peroxidase activity assay, the result showed the
highest MIP activity after 2 weeks of cultivation. After that, the activity
was static or decreases (Fig. 1c). It was found that in the
presence of CuSO4, MIP activity was higher than that of control.
However, in the presence of CuSO4 (0.04-2.4 μmole g-1
solid substrate) showed no difference in MIP activity and found that at 0.04
μmole CuSO4 the highest MIP activity was observed. Figure
1d represents MnP activity increasing according to CuSO4 concentration.
However, in the presence of CuSO4 (0.16-2.4 μmole g-1
solid substrate) showed no difference in MnP activity. When considered the MnP
specific activity, it also showed consistent results (data not shown). The major
ligninolytic enzymes produced by the fungus were laccase and followed with MIP
and MnP, respectively with no LiP activity have been confirmed in this study.
Similar observation in the culture on rice husk and bran without inducer previously
reported (Sarnthima et al., 2009).
Extracellular carbohydrate degrading enzymes production: L. polychrous
has not yet been reported whether it can produce and secrete carbohydrate-degrading
enzymes, such as xylanase, cellulase and β-glucosidase. Thus, it is necessary
to measure these enzyme activities if application of paper and pulp industry
is in consideration which is because these enzymes have certain effects on pulp
and paper quality. The results showed that the crude extract had no xylanase
activity, while β-glucosidase and cellulase activities had been found in
low quantities (Fig. 2a, b). The fungus
gave very low cellulase activity when compared to T. versicolor, Phellinus
sp., Daedalea sp. and P. coccineus (Liew
et al., 2010). This low carbohydrate-degrading activity further suggests
that this white rot strain is more interested applying for pulp biobleaching.
Take a close look at culture of 28 days; CuSO4 could induce ligninolytic
enzymes activity as shown in Fig. 3a and carbohydrate degrading
enzymes activity as shown in Fig. 3b. From the results, we
chose 0.8 μmole CuSO4 per gram solid substrate for further experiment
to produce a large quantity of laccase. This culture condition showed activity
ratios of laccase to peroxidase, β-glucosidase to cellulases and ligninolytic
enzymes to carbohydrate degrading enzymes were 2.0, 1.6 and 74.0, respectively
|| β-glucosidase (a) and Cellulase activity and (b) in
the absence and presence of 0.04, 0.16, 0.8 and 2.4 μmole CuSO4
per gram solid substrate at different times of cultivation
|| Ligninolytic enzymes (a) carbohydrate degrading enzymes,
(b) production by L. polychrous grown for 28 days on solid-state
fermentation with various concentrations of CuSO4 and comparison
in enzyme ratios of 0.8 μmole CuSO4 per gram solid substrate
and (c) Activity of Lcc, laccase; Pox, peroxidase; β-gluc, β-glucosidase;
Ligno, ligninolytic enzyme; CHO, carbohydrate degrading enzyme
|| pH optimum (a) and temperature optimum (b) of crude laccase
from L. polychrous grown on solid-state fermentation with 0.8 μmole
CuSO4 per gram solid substrate
Some white rot fungi secrete carbohydrate-degrading enzymes, apart from ligninolytic
enzymes during lignin degradation in nature including xylanase, cellulase and
glucosidase (Leonowicz et al., 1999; Quiroz-Castaneda
et al., 2009).
Enzyme characteristics: The crude enzyme of L. polychrous from
SSF supplemented with 0.8 μmole CuSO4 per gram solid substrate
was studied its optimize condition for catalysis. The results showed that
the optimum pH for laccase catalysis was in the range of 3-4 (Fig.
4a) and optimum temperature was at 55°C (Fig. 4b).
The pH and thermal stability of crude laccase of this fungus were also experimented.
The crude laccase was well stabilized in distilled water as well as in buffers
pH 6-8 (approximately 90 and 80% residual activity at 24 h compared to the original
|| pH stability (a) and thermal stability, (b) of crude laccase
from L. polychrous grown on solid-state fermentation with 0.8 μmole
CuSO4 per gram solid substrate
The thermal stability of the crude laccase, when the temperature is high, crude
laccase tended to denature. The crude laccase was well stabilized at 30°C
in 0.1 M sodium acetate buffer, pH 4.5 with residual activity more than 80%
after 24 h incubation (Fig. 5b). Moreover, at temperature
of 40°C, laccase could well stabilize for several hours, even though its
activity was decreased to about 70% residual activity at 5 h incubation but
at 24 h its activity was about 60% left. Even though its optimum temperature
for catalysis seemed as high as 50-55°C (Fig. 4b), in
this temperature the enzyme lost its activity quickly and completely inactive
at 2-3 h incubation (Fig. 5b). These aspects of the enzymes
from this fungus are important to consider if temperature is a key in industrial
process in order to optimize the application of enzyme biocatalysts.
Electrophoresis of the crude enzyme in native condition revealed that there
are at least two isoenzymes (or isoforms) of laccases as it appeared two bands
of activity stains with laccase substrates ABTS and syringaldazine (Fig.
6 lane 1, 2). Interestingly, those bands with laccase activities also stained
with peroxidase substrates (Fig. 6 lane 3, 4). These results
were agreed with previous report of crude enzyme of L. polychrous culture
in solid state of rice husk and bran without CuSO4 inducer by Sarnthima
et al. (2009) in which more than one laccase isoenzymes (or isoforms)
observed. However, instead of three bands of activity zymogram, only two bands
observed here in this work. These results indicate that CuSO4 have
certain effect on extracellular enzyme production and secretion by this fungus.
The protein bands of coomassie staining showed more than two bands by two of
them were at the same electrophoretic mobility corresponding to extracellular
enzymes (Fig. 6 lane 5).
||Native-PAGE of crude laccase from L. polychrous grown
on solid-state fermentation with 0.8 μmole CuSO4 per gram
solid substrate, stained with substrates of ligninolytic enzymes and with
coomassie brilliant blue R-250. 1: with ABTS, 2: with Syringaldazine, 3:
MBTH+DMAB+Mg2++ H2O2, 4: Syringaldazine
followed with MBTH+ DMAB+Mg2++H2O2, 5:
with coomassie brilliant blue R-250. Arrows indicate expected ligninolytic
It seemed that the fungus secrete proteins mainly ligninolytic enzymes with
low amount of carbohydrate degrading enzymes which might be other two bands
that not corresponding with ligninolytic activities (highest and lowest electrophoretic
mobility). If possible, activity stain with β-glucosidase and cellulase
substrates might reveal more insight information of the crude enzyme of this
fungus. Moreover, successful purification of those enzymes would be very useful
in order to further study about their properties, structure and function relationship.
Versatile peroxidases are hybrids of lignin peroxidase and manganese peroxidase
with a bifunctional characteristic (Wong, 2009).
Black liquor decolorization: Dark colors in pulp and paper mill effluent come from lignin and its derivatives. Lignin compounds are polymer substances which complex in structure and not simple to degrade. Since the fungi, especially the white rot fungi are reported about their ability to degrade lignin in nature, because they can produce and secrete ligninolytic enzymes for lignin degradation and use as a carbon source. Lininolytic enzymes from L. polychrous mainly are laccase and MnP but no LiP activity could be detected. They have potential uses in many areas, especially dye decolorization. Therefore, the crude laccase was tested for black liquor decolourization.
To evaluate the potential application of the crude enzyme from L. polychrous
for black liquor decolorization, reactions were done at various pH conditions
(pH 3-9) using laccase activity 0.2 U mL-1 in the absence and presence
of ABTS and HBT redox mediators (0.05 mM) at 32°C for 24 h. The results
are as shown in Fig. 7a-c. In the absence
of redox mediator and in the presence of ABTS, only about 10% black liquor decolorization
in buffer pH 8.0 could be observed at 24 h. Black liquor decolorization have
been reported by some other white rot fungi such as Cyathus bulleri and
others accompanying with certain redox mediators (Da Re
and Papinutti, 2011).
Black liquor decolorization at pH 3 occurred precipitation of lignin in reactions
in all tested condition. At this pH, in the absence of ABTS redox mediator showed
highest percentage decolorization of 52.2% at 5 h (Fig. 7a),
while in the presence of ABTS gave the highest percentage decolorization of
56.8% at 5 h (Fig. 7b) and about 50% at 24 h in the presence
of HBT redox mediator (Fig. 7c). At the rest pH conditions
the black liquor reactions increased in its maximum absorbance (minus graphs),
suggesting that polymerization might occurred instead of decolorization (Fig.
During black liquor decolorization reactions, the residual activity of laccase
was monitoring in each reaction tube at different times. In the experiment,
both absence and presence of ABTS or HBT redox mediators, the laccase activity
tended to decrease when the time progresses (Fig. 8a-c).
The crude laccase was well stabilized in pH 4-6 whereas in other pH conditions,
laccase activity clearly decreased with reaction time.
The crude enzyme seemed to not able to decolorize black liquor even in the
presence of ABTS or HBT redox mediator even though the crude laccase fairly
stable throughout experimental time scales. However, at pH 3 it introduced a
lignin precipitation and made paler black liquor where the laccase was least
stable. Lignin could precipitate in acid condition, at pH 3 was also observed
lignin precipitation in this work after stand for 24 h. However, in enzymatic
reaction of the crude enzyme such precipitation could be observed within 3 h.
This result suggest that reaction of lignin by the crude enzyme might accelerate
precipitation of lignin compounds in the black liquor.
|| Black liquor decolorization in various pH conditions in the
absence of redox mediator (a) in the presence of redox mediator, 0.05 mM
ABTS (b) and 0.05 mM HBT (c)
|| Remaining laccase activity in reactions of black liquor decolorization
in various pH conditions in the absence of redox mediator (a) in the presence
of redox mediator, 0.05 mM ABTS (b) and 0.05 mM HBT (c)
This might be applied crude enzyme of the fungus for recovery of lignin for
another purpose use instead of concentrate acid precipitation. Lignin precipitation
by acid has been reported in many works (Koljonen et
al., 2004; Fernando et al., 2010). Today,
lignin extraction from black liquor is an interesting option for pulp mills
due to possibility to increase the production capacity of pulp without increasing
the load in the recovery boiler. Moreover, extracted lignin can be used as a
solid biofuel or as a feed stock producing various chemicals. Application of
crude laccase of L. Polychrous in acidic condition might shorten the
precipitation process and lower use of concentrate acid.
Lentinus polychrous produced ligninolytic enzymes including laccase, MIP and MnP but no LiP activities. Copper(II)sulfate at low concentration could induce laccase production and affected isoenzyme pattern. No xylanase activity but low cellulase and β-glucosidase activities, fairly high optimum temperature and thermal stability as well as be able to work in pH 6-8 suggested the crude ligninolytic enzyme from this fungus a promising potential to apply for lignin removal in pulp biobleaching or treatment of wastewater.
The authors would like to thank the Thailand Research Fund Master Research Grant No. MRG-WI525S104 as well as the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Department of Chemistry, Faculty of Science, Mahasarakham University. Many thanks also go to the Phoenix Pulp and Paper Public Company Limited for a partial financial support.