Isolation of laccase producing fungi and partial characterization of

laccase
Savitha S. Desai, Gururaj B. Tennali, Nityanand Channur, A.C. Anup, Gouri
Deshpande, B.P. Azhar Murtuza

Department of Biotechnology, B.V.B. College of Engineering and Technology, Hubli, Karnataka

580031, India; Email: joshisavitha@bvb.edu

ABSTRACT
Laccase is a copper-containing polyphenol oxidase that acts on a wide range of substrates. This enzyme is
found in many plant species and is widely distributed in fungi including wood-rot fungi, where it is often
associated with lignin peroxidase, manganese dependent peroxidase, or both. Laccases have widespread
applications, ranging from effluent decolouration and detoxification to pulp bleaching, removal of phenolics
from wines and dye transfer blocking functions in detergents and washing powders. The biotechnological
application of laccase has been expanded by the introduction of laccase- mediator systems, which are able to
oxidize non-phenolic compounds that are otherwise not attacked and are thus able to degrade lignin in kraft
pulps. Hence, the present research work was carried out to isolate potential laccase source and partially
characterize the laccase enzyme. Soil and tree bark scrapings from farms and forests of different places in and
around Hubli were screened for isolation of laccase producing fungi. Fungi were cultivated on potato dextrose
agar (PDA) plates containing indicator compounds namely guaiacol and tannic acid. It resulted in isolation of
9 fungal strains, among which one of the strains was presumed to be potent depending on its growth
characteristics and was used for further experiments. It was identified as belonging to Tricoderma genus.
Higher level of laccase activity was observed under solid state condition production (15.7 U/ml) than in
submerged condition (10.68 U/ml). It was also found out that the laccase was active over broad range of
temperature and pH, with optimum temperature of 45ºC and pH 5. The isolated strain can be further worked
for other characteristics and it could prove to be a potent enzyme for lignin degradation.

Keywords: laccase, lignin proxidase, manganese dependent peroxidase, tannic acid, guaiacol, Trichoderma

INTRODUCTION

Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are multi-copper enzymes belonging

to the group of blue oxidases. They are defined as oxidoreductases, which oxidizes diphenol and
allied substances. Molecular oxygen serves as the terminal electron acceptor and is thus reduced to
two molecules of water [1]. The ability of laccases to oxidize phenolic compounds as well as their
ability to reduce molecular oxygen to water has led to intensive studies of these enzymes [2-4].
Laccases are typically found in plants and fungi. Plant laccases participate in the radical-based
mechanisms of lignin polymer formation [5-9], whereas in fungi laccases probably have more roles
including morphogenesis, fungal plant-pathogen/host interaction and stress defense and lignin
degradation [4]. Although there are also some reports about laccase activity in bacteria [10-13], it
does not seem probable that laccases are common enzymes from certain prokaryotic groups. Most
of the laccases reported thus far are of fungal origin, especially from white rot fungi [14] such as
Phlebia radiata, Pleurotus ostreatus and Trametes versicolor [15].
Discovery of novel laccases with different substrate specificities and improved stabilities is
very important for industrial applications, besides developing an effective high yield and economic
production medium enhances its utility. Several reports regarding this aspect reported thus far have
Research Article, Biotechnol. Bioinf. Bioeng. 2011, 1(4):543-549
© 2011 Society for Applied Biotechnology. Printed in India; ISSN 2249-9075
544

been worked on both submerged and solid state fermentation. Solid state fermentation utilizing
natural lignin containing substrates such as rice bran, wheat bran, coir dust, potato peel, etc. [16,17]
have received much attention because of its efficiency in production of microbial laccases. The
enzymatic catalysis by laccases in different industrial applications such as textile dye bleaching,
pulp bleaching and bioremediation could serve as a more environmentally benign alternative than
the currently used chemical processes. Hence, the present research work aimed at isolation of potent
laccase producing fungi, establishment and comparison of submerged and solid state conditions for
laccase production and partial characterization of the enzyme extracted from potent strain.

MATERIALS AND METHODS

Isolation of laccase producing microorganisms

Soil samples and tree bark scrapings, collected from forests of Yellapur, Kiruvatti, Tadas and
Ankola in Karnataka, India were for isolation by employing standard serial dilution method. The
laccase producing fungi were screened based on the growth on potato dextrose agar (PDA) media
containing specific substrate of laccase which included tannic acid and Guaiacol [14]. PDA plates
were observed for growth and development of brown colored precipitate in tannic acid containing
plates and reddish hallow zone in guaiacol containing plates.

Selection of potent strain and identification

All the isolated organisms were inoculated on Petri plates containing PDA medium with tannic acid
and observed for development of brown colored precipitate and was assessed on daily basis. The
organism showing faster growth was selected as potent strain. The strain was identified by
Lactophenol dye method and observed under microscope [18].

Selection of production medium

Submerged condition

Potent strain was cultivated in Olga liquid medium containing 3g peptone,10g glucose,0.6gm
KH2PO4,0.001g ZnSO4, 0.4g K2HPO4, 0.0005g FeSO4, 0.05g MnSO4 and 0.5g MgSO4 per L, and
kept in incubator shaker at 200 rpm, 30ºC for 12 days. 3ml of broth was collected from the fourth
day, fungal mycelium was separated from the broth by filtering it through Whatman No. 1 filter
paper. The filtrate collected was used for enzyme assay and all the experiment was carried out in
duplicates [19].

Solid state fermentation

The solid state production was carried out using wheat and rice bran obtained from the local market.
Six Petri plates containing 5g wheat bran moistened with distilled water (1:1 w/v) were incubated
with selected organism at 30ºC in an incubator for 8 days. Autoclaved water (12 ml) was dispensed
in to a PDA slant (incubated with selected organism at 30ºC for 7days) properly mixed and equal
amount of water was then used for inoculating individual plate. The enzyme activity was measured
from the fourth day of incubation. Crude culture filtrate was obtained by adding 15ml of distilled
water to the plates and filtering through muslin cloth. It was further centrifuged at 10000 rpm for 10
min. The supernatant was used for enzyme assay [20]. Similar procedure was carried out for rice
bran, experiments for both substrates were carried out simultaneously and activity levels were
compared.
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Laboratory scale production on rice bran

The organism yielded a higher enzyme activity on rice bran, than with wheat bran and afore
mentioned liquid medium. 5ml of autoclaved water dispensed in to PDA slant incubated at 30ºC
with selected organism was used for inoculating 500 ml conical flask containing 50g rice bran
moistened with distilled water (1:1 w/v), flask was sterilized in autoclave at 121ºC for 20min before
inoculation. Crude filtrate was obtained by adding 100ml of distilled water to the flask and filtering
through muslin cloth. The filtrate was again centrifuged at 10000 rpm for 10 min. The supernatant
collected was further used as enzyme source.

Guaiacol assay method for laccase assay

Guaiacol has been reported as efficient substrate for laccase assay. The intense brown color
development due to oxidation of guaiacol by laccase can be correlated to its activity often read at
450 nm. Guaiacol (2mM) in sodium acetate buffer (10mM pH 5.0) was used as substrate. The
reaction mixture contained 3ml acetate buffer, 1ml guaiacol and 1ml enzyme source and enzyme
blank contained 1ml of distilled water instead of enzyme source. The mixture was incubated at 30ºC
for 15min and absorbance was read at 450nm blank using UV spectrophotometer [21]. Enzyme
activity was expressed as International Units (IU), where 1 IU is defined as amount of enzyme
required to oxidize 1micromole of guaiacol per min. The laccase activity in U/ml is calculated using
the extinction coefficient of guaiacol (12,100 M-1 cm-1) at 450 nm by the formula: E.A = (A* V) / (t
* e * v), where E.A = Enzyme Activity (U/ml), A = Absorbance at 450nm, V = Total volume of
reaction mixture (ml), v = enzyme volume (ml), t = Incubation time (min) and e = Extinction
Coefficient (M-1cm-1).

Characterization of enzyme

The effect of temperature on laccase activity was determined by recording the absorbance of
enzyme catalyzed reaction using guaiacol (2mM) as substrate, dissolved in sodium acetate buffer
(10mM pH 5.0), incubated at temperature 15, 25, 35, 45, 55 and 65ºC. The reaction mixture was
incubated for 15min. Temperature at which enzyme showed maximum activity, was noted as
optimum temperature of enzyme. The influence of pH on laccase activity was studied by recording
the absorbance of enzyme catalyzed reaction at optimum temperature, using guaiacol (2mM) as
substrate dissolved in buffers of different pH (acetate buffer pH 4, pH 5, phosphate buffer pH 6, pH
7 and tris-glycine buffer pH 8) and incubated at 25ºC for 15min and absorbance were recorded at
540nm.

RESULTS AND DISCUSSION

Screening of samples for laccase producing fungi

Total of nine organisms were isolated from samples as laccase producers, as all of them oxidized
tannic acid present in the screening medium (Table 1). It was also identified by development of dark
brown precipitate below the colonies. The incubation period required for proper growth and
development of colored precipitate varied with individual organism. Rich supplements of laccase
specific substrates in the forest and farm soil and tree barks make them rich habitats for laccase
producing fungi.
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Table 1. Isolated strain, sample type and their locations.

Strain code Type of Sample Location

LS1 Farm Soil Tadas, Hubli
LS2 Farm Soil Tadas, Hubli
LS3 Forest Soil Kiruvatti, Yellapur
LS4 Forest Soil Kiruvatti, Yellapur
LB1 Bark Scraping Ankola
LB2 Bark Scraping Shirle, Yellapur
LB3 Bark Scraping Shirle, Yellapur
LB4 Bark Scraping Kolikere
LB5 Bark Scraping Kolikere

Selection of potent strain

It was observed that, LS1 strain, isolated from the farm soil sample showed fast growth and brown
color precipitate within 3 days of incubation period. Other strains took more than 6-7 days of
incubation for appropriate growth and oxidation of substrate employed in the medium. Thus strain
LS1 was presumed to be the potent among all isolates and hence was used for further work. The
selected LS1 strain was identified based on its colony morphology as well as microscopic
visualizations (spore and hyphae). The organism produced white to green cushions of sporulating
filaments (Figure 1). The microscopic observations showed the arrangements of conidia in balls at
the tips of filaments. These colony characteristics and microscopic visualizations were similar to the
fungi genus Trichoderma [18]; hence the isolated strain was concluded to be a member of
Trichoderma genus. T. atroviride, T. longibrachiatum, T. harzianum are some of those Trichoderma
spp. studied as laccase sources.

Figure 1. Colony morphology of isolated strain, LS1; white mycelia growth with green
spores can be visualized.

Production of laccase in submerged condition

The potent organism was initially cultivated in medium of Olga [19], which is a high laccase
yielding medium. The enzyme activity was measured every day after the third day of incubation and
continued until decrease in activity level was observed. The enzyme activity was high on the 6th day
 547

of incubation (Figure 2). The level of laccase activity was comparatively low (10.68 U/ ml) due to
lack of certain inducers of laccase.

Production of laccase under solid state condition

It has been reported that fungi grow at faster rate and produces good biomass on solid substrates
[23] than in submerged conditions. The organism selected as potent one showed both faster growth
and good amount of biomass. It was cultivated on two different natural substrates, wheat bran and
rice bran. The organism though showed comparable growth pattern on both solid media, maximum
activity was read on 6th day of incubation. The enzyme activity of organism grown on rice bran was
higher (15.7 U/ml) than on wheat bran (11.59 U/ml; Figure 3). It was observed that fungal laccase
activities in extracts of solid media were higher when compared with those in extracts of liquid
media. This result may be related to i) lower deactivation of enzymes after adsorption or
immobilization on agricultural residues in solid media, and/or ii) more stimulation and production
of enzymes in solid media [23].

Figure 2. Graph displaying the activity of enzyme produced after days of incubation under
submerged condition [activity (U/ml) v/s incubation time (days)].

Characterization of the enzyme

The optimum temperature laccase obtained from the organism was found to be 45ºC (Figure 4). The
temperature range where the enzyme is active is remarkably wide. Significant activity is detected as
low as 15ºC and approximately 95% of activity is maintained at temperatures as high as 60ºC.
Optimum temperatures are significantly affected by the assay used so this data should be interpreted
with care. According to literature the typical optimum temperature range for laccases is 50ºC to 60
ºC [24]. Laccase enzyme extracted from Trametes versicolor exhibited high enzyme activity at a
temperature of 50ºC [25]. Laccase purified from Stereum ostrea found to be active and stable at
temperature 40ºC [25].
The normal range of pH for typical laccase enzyme is 3-5. It has been also reported that
optimum pH varies with type of substrate employed [26]. Laccase from the LS1 had pH optima of 5
with guaiacol as substrate [Figure 5]. The optimum pH value for laccases varies depending on the
substrates employed, even though many reports have been reported a bell shaped profile for laccase
activity. pH optima vary considerably due to reactions caused by substrate utilized, molecular
oxygen or enzyme itself. The difference in redox potential between the phenolic substrate and the
T1 copper could increase oxidation of the substrate at high pH values, but the hydroxide anion OH-
_ binding to the T2/T3 copper results in an inhibition of laccase activity due to disruption of the
internal electron transfer between T1 and T2/T3 Centers. These two opposing effect can play an
important role in determining the optimal pH of the biphasic laccase enzyme. Laccase enzyme
548

Figure 3. Graph displaying the activity of enzyme produced after particular days of incubation
under solid state condition; the activity observed under both Wheat Bran (WB) and Rice Bran
(RB) is compared [activity (U/ml) v/s incubation time (days)].

Figure 4. Graph displaying the activity of the enzyme at particular temperature; the enzyme
activity reached the maximum at particular temperature and decreased thereafter [activity
(U/ml) v/s temperature (ºC)].

Figure 5. Graph displaying the activity of the enzyme at particular pH; the enzyme activity
reached the maximum at particular pH and decreased thereafter [activity (U/ml) v/s pH].
 549

extracted from Trametes versicolor [24] exhibited high enzyme activity over broad pH and
temperature ranges with optimum activity at pH 3.0 and temperature of 50ºC. Laccase purified from
Stereum ostrea found to be active and stable at optimal pH 6.0 and temperature 40ºC [25].

CONCLUSION

Screening for laccase producing fungi on plates containing colored indicators resulted in isolation of
nine different fungal strains. One of the strains was identified as potent due to its fast growth rate
and grater reactivity towards colored indicator present in the cultivating medium and
microscopically identified as belonging to Trichoderma genus. Laccase production by isolated
Trichoderma strain was carried out both in submerged and solid state condition. The production
level of laccase in submerged condition was quite low (10.6 U/ml) in medium of Olga, whereas in
solid state condition employing wheat bran and rice bran, resulted adequate levels of laccase yields.
Rice bran yielded higher levels of laccase (15.7 U/ml) than wheat bran (11.59 U/ ml). The crude
laccase was found to have maximum activity at 45ºC and at pH 5. This potent organism can be used
for large scale laccase production and its use in treating various industrial effluents.

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