Indian Journal of Biotechnology

Vol. 17, January 2018, pp 33-43

Identification of efficient dye decolorizing laccase

producing fungi from Kolli Hills
Periasamy Rathinasamy1* and Palvannan Thayumanavan2
1
Department of Public Health, Aksum University, Ethiopia
2
Department of Biochemistry, Periyar University, Salem 636011, Tamilnadu, India

Received 6 November 2013; revised 4 April 2016; accepted 21 April 2016

Laccase is one of the most promising ligninolytic enzymes for the industrial application and ecofriendly bioremediation
process. Twenty-five carpophores were collected from different places of Kolli Hills (Namakkal district Tamilnadu, India)
and screened on the solid media containing guaiacol, which enabled the detection of laccase secretion. Three positive strains
were isolated and the quantitative production of laccase was determined in submerged culture to select hypersecretory strain
for further study. Among the three strains, “ST02” the best producer of laccase was selected and was analyzed for the dye
decolorization potential using dyes like Poly R-478 and Remazol Brilliant Blue R (RBBR). Identification of the isolated
organism was carried out by classical and molecular methods. Approximately 625 bp of the ST02 5.8S rDNA was amplified
by polymerase chain reaction (PCR). The phylogenetic relationship of the isolated strain was studied by comparing the
internal transcribed spacer (ITS) sequences of ST02 with similar related sequences deposited in the GenBank database. The
present study showed that relatively simple plate test screening method and ITS analysis can be used for identification of
laccase producing new strain. The isolated organism was designated as Pleurotus ostreatus IMI 395545.

Keywords: Laccase, Pleurotus ostreatus IMI 395545, internal transcribed spacer, guaiacol, phylogenetic analysis

Introduction production6-8 or with liquid cultivation monitored with

Laccases are interesting enzymes for industrial enzyme activity measurements9. As laccases oxidize
applications because extensive studies have shown the various types of substrates, several different
potential of fungal phenol oxidases as a biological compounds have been used as indicators for laccase
alternative for chemical oxidative processes e.g. production. Recently, Chhaya and Gupte (2010)10
pulp delignification in textile industry, food industry, identified a new strain Fusarium incarnatum for the
bioremediation, organic synthesis, pharmaceutical production of laccase using o-dianisidine in the
sector and nanobiotechnology1. Recently, most plate test. Inadequate experimentation may lead to the
of the laccase studied are of fungal origin, especially misidentification of a laccase for other phenol
from white rot fungi, Anthracophyllum discolor2, oxidizing enzymes such as manganese peroxidase.
Pycnoporus sanguineus3, Trichoderma harzianum4 The use of substrates such as syringaldazine
etc. Screening of a large number of white-rot fungi is, and ABTS [2,2-azino-bis(3-ethylbenzthiazoline-6-
therefore, necessary to select strains that are able to sulfonic acid)] should be used with caution since these
produce high titers of laccases with novel characters. substrates are catalyzed by both laccase and
Such a screening trial should preferably rely on the manganese peroxidase11. Plate test screening with
use of inexpensive, rapid and sensitive testing laccase indicator compounds has been reported by
methods and the screening strategy must be compiled many groups12-15.
in such a manner as to identify fungal strains and In fungal taxonomy, sequences from the internal
enzymes that will work under industrial conditions5. transcribed spacers (ITS) region of the nuclear rDNA
Microbes that produce laccases have been screened are commonly used for the identification of fungi16-18.
for either on solid media containing colored indicator The ITS sequence including both ITS1 and ITS2,
compounds that enable the visual detection of laccase which are separated by the conserved short 5.8S
rRNA, has been commonly used to infer phylogenetic
———————
*Author for correspondence:
relationships of closely related species as well as
Mobile: +251-974308602; +91-8903793546 to assess the variability of a population, e.g. of
[email protected]; [email protected] geographically distant isolates (ecotypes). Since the
34 INDIAN J BIOTECHNOL, JANUARY 2018

ITS region is highly conserved intra-species level but presence of brick red color around the mycelium was
variable between different species it is often used in considered as guaiacol oxidizing laccase secreting
taxonomy19. The evolutionary distance is generally organism.
displayed in the form of trees and a wide diversity
Laccase Production in Liquid Media
of algorithms was available to construct them.
The selected three positive fungal strains detected
Innovation of novel laccases with different substrate
in the plate test, were subjected to qualitative
specificities and improved physical properties such as
determination of laccase production in submerged
thermostable or acid tolerant is imperative qualities
culture20. The strains were grown in 50 ml of 4%
for industrial applications.
potato dextrose broth (PDB) in a 100 ml Erlenmeyer
In this study, laccase-producing fungi were isolated
from various samples around the Eastern Ghats of flask. The flasks were incubated at 30C on a rotary
Kolli hills. In addition, the production of laccase by shaker (120 rpm).
the positive strains was confirmed in liquid culture. Dye Decolorization Potential
The isolated hypersecretory strain was identified by Dye decolorization potential of the isolated
phylogenetic analysis. hypersecretory strain was carried out according to the
method of Kiiskinen et al22. In brief 0.04% (w/v) Poly
Materials and Methods R-478 or RBBR was added individually to 15 ml of
4% PDA in Petri plates. The pH was adjusted to 5.5
Chemicals before autoclaving at 121°C for 15 min. The dyes
Catalase, lactophenol, cotton blue and potato were added to the media after autoclaved as sterile
dextrose agar (PDA) were purchased from HiMedia, filtered water solutions. The positive reaction on the
(Mumbai, India). Chloramphenicol, Poly R-478 and plate was visualized within 5-6 d of incubation at
Remazol Brilliant Blue R (RBBR) were purchased 30°C.
from s d fine-chem Limited, India. Benomyl (Benofit)
was purchased from Coromondel Fertilizers Limited Enzyme Assay
(Tamil Nadu, India). SoluteReady® genomic DNA Laccase activity was determined using guaiacol as
purification kit, PCR reagents, agarose gel the substrate according to the method of Sandhu and
electrophoresis consumables and primers were Arora23. The assay mixture contained 4.80 ml of
purchased from Helini Biomolecules (Chennai, India). sodium phosphate buffer at pH 6.0 (100 mM), 0.1 ml
of guaiacol (10 mM) and 0.1 ml of enzyme extract.
Isolation of Microorganism One activity unit (U) was defined as the amount of
Twenty-five basidiomycetous carpophores (stalk of enzyme oxidizing 1 μmol guaiacol per minute. The
a fruiting body in fungi) were collected from the kinetic reaction was spectrophotometrically recorded
deep forest of Kolli Hills in Namakkal district, at 470 nm (ε = 21,600/M/cm) incubated at 60°C
Tamil Nadu, India in early rainy season in the year for 30 min, in an UV-Visible Spectrophotometer
2008. Carpophores were picked and stored in 118 (Systronics, India). The blank contained all the
thermocol boxes. The samples were then sprayed with assay constituents except the active enzyme, buffer or
70% ethanol and soaked in Petri dishes for 5 minutes heat inactivated enzyme was used in its place. The
for surface sterilization. A sample of the interior flesh enzyme activity was expressed as U/l.
of the fruiting body was excised and placed on the In order to rule out the role of peroxidases in the
PDA to isolate the fungi20. In addition, 0.01% (w/v) oxidation and prove the oxidation was only by
chloramphenicol or chlorotetracycline was added to laccase, the enzyme was pre-incubated with catalase
the media in order to inhibit the growth of bacteria (1000 units/ml) (EC: 1.11.1.6) for 30 min at 30C
and 1% (w/v) of benomyl was added in order to select prior to assay to remove any endogenous hydrogen
for wood decay fungi21. peroxide24. Lignin peroxidase (LiP) and manganese
Screening Test dependent peroxidase activities (MnP) were measured
The ability of the fungal strains to secrete as follows:
extracellular laccase was visualized according to the Lignin peroxidase (LiP), (EC 1.11.1.14) activity
method of Kiiskinen et al22. The assay plate contained was determined by monitoring the oxidation of
15 ml of 4% PDA amended with 0.01% of guaiacol. veratryl alcohol to veratraldehyde (ε = 9,300/M/cm) at
The plates were incubated at 30C for 1–3 days. The 37°C as indicated by an increase in absorbance at 310
 RATHINASAMY & THAYUMANAVAN: DYE DECOLORIZING LACCASE PRODUCING FUNGI FROM KOLLI HILLS 35

nm25. The reaction mixture (2.5 ml) contained 0.5 ml 1, 5.8 S rDNA, ITS 2 and the 5’ end of 28 S rDNA
enzyme extract, 0.5 ml H2O2 (2 mM), 0.5 ml veratryl were amplified using primers ITS1 (5´TCCGT
alcohol solution (10 mM) and 1.0 ml sodium acetate AGGTGAACCTGCGG3´) and ITS 4 (5´TCCT
buffer pH 3.0 (10 mM). One unit of enzyme activity CCGCTTATTGATATGC3´)29. PCR amplification
was defined as the amount of enzyme oxidizing was performed in a total volume of 25 μl
1 μmol of substrate per minute. The enzyme activity amplification reactions contained 10X Taq buffer,
was expressed as U/l. 2 mM MgCl2, 0.4 mM dNTP mix, and 2 U
Manganese peroxidase (MnP), (EC 1.11.1.13) proofreading Taq DNA polymerase. One micro litre
activity was measured with phenol red as the substrate of both forward and reverse primers (10 pmoles/μl)
at 610 nm26. Reaction mixture contained 0.5 ml were added with 2 l of genomic DNA. The volume
enzyme extract, 0.1 ml phenol red solution (0.1%), of the mixture was made up to 50 l by adding 21 l
0.1 ml sodium lactate pH 4.5 (250 mM), 0.2 ml of nuclease free water. The mixture was mixed gently
bovine serum albumin solution (0.5%), 0.05 ml and spun down briefly for about 30 min. PCR was
manganese sulphate (2 mM) and 0.05 ml H2O2 performed in a thermocycler (Corbett Research,
(2 mM) in sodium succinate buffer pH 4.5 (20 mM). Austria). Initial denaturation of 95°C for 2 min was
Activity was expressed as increase in absorbance at followed by 30 cycles of 94°C for 1 min, 58°C for
610 nm per minute per milliliter. One unit of enzyme 1 min (primer annealing), 72°C for 1 min (primer
activity was defined as the amount of enzyme extension). A final extension of 72°C for 5 min
oxidizing 1 μmol of substrate per minute. The enzyme was incorporated, followed by cooling to 4°C until
activity was expressed as U/l. recovery of the samples. Sequencing of the PCR
Fungal Identification Method products was carried out by Helini Biomolecules
The classical method of identifying fungi is by (Chennai, India) on an ABI Prism 3100-Avant
using light microscopy. The spore structure of the Genetic Analyzer (USA) and the obtained sequence
fungi was identified by lactophenol cotton blue was aligned using the BioEdit sequence alignment
mounting method27. editor version 7.0.4.1.
Isolation of Genomic DNA Analysis of PCR Product
Twenty grams of mycelium was used for DNA Amplified PCR products were electrophoresed in a
isolation according to Hamelin et al28, with some 2% agarose gel using 1X Tris–borate–EDTA buffer
modifications. The fungal mycelium grown on PDB (100 mM Tris–HCl/l, pH 8.3, 83 mM boric acid/l, 1
was harvested and any media attached to it was mM EDTA/l) at 100 V and visualized by ethidium
removed by washing with sterile water. The washed bromide staining. Sizes of the amplified products
sample was ground to a fine powder in liquid were determined using different size of standard DNA
nitrogen. 15 ml of extraction buffer (1.4 M NaCl, ladder. The reproducibility of DNA profiles was
0.1 M Tris-HCl (pH 8), 0.02 M EDTA, 200 μl tested by repeating twice the PCR amplification with
β-2-mercaptoethanol, 2% cetyl-trimethyl-ammonium each of the selected primers. Only reproducible bands
bromide) was added to 100 mg of the grounded were considered for analyses.
mycelia. The mixture was incubated at 65°C for Analysis of Sequence Data
1 h and extracted by adding 10 ml of phenol: A comparative study has been carried out with
chloroform: isoamyl alcohol (25:24:1). This was other rDNA sequences with the rDNA from the
gently shaken and centrifuged at 15,000 rpm. selected fungus (ST02) was accomplished using the
Supernatant was transferred to a new tube and the BLAST30 (http://www.ncbi.nlm.nih.gov). Thirty-four
aqueous phase was precipitated with cold isopropanol most similar meaningful sequences related to the
by incubating for 1 h at −20°C, and then centrifuged isolated fungal sequence were downloaded and
for 15 min at 15,000 rpm. The pellets were washed analyzed. Related fungal sequences were selected by
with 70% ethanol, air-dried, resuspended in 50 μl of basic local alignment search tool (BLAST) match and
TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) then by FASTA query of the National Centre for
and stored at 4°C. Biotechnology Information (NCBI) fungi data subset.
PCR Targeting ITS Region and Sequencing the Amplicon The selected sequences were aligned by using
From the total genomic DNA, a DNA segment clustalW231 version CLUSTAL 2.0.12. All the
containing the 3’ end of the nuclear 18 S rDNA, ITS sequences were used to construct the phylogenetic
36 INDIAN J BIOTECHNOL, JANUARY 2018

tree by using Geneious pro trial 4.8.5 software with shows the production of laccase by selected strains.
the following parameters i.e. 65% similarity (5.0/-4.0) Among the three strains, ST02 which showed highest
cost matrix, Gap open penalty = 12, Gap extension production of laccase (410 ± 1.6 U/l) was selected for
penalty = 3. The construction of phylogenetic tree was the further analysis. The selected hypersecretory
based on the Tamura et al32 genetic distance model. positive strain ST02 was subjected to decolorization
The alignment was inspected closely to ensure that of indicator dye compounds. Figures 3a & b displays
were no PCR chimaera events. qualitative decolorization of Poly R-478 dye on
incubation with ST02 strain. Initial pink color of the
Results dye was decolorized to colorless on the 6 days of
Among the 25 samples collected, 3 strains were incubation with ST02. Figures 3c & d shows the
selected as positive organisms which formed halo of RBBR dye was completely decolorized on 6 days of
intense brown color around the fungal colonies. Based incubation. In the case of RBBR, initial blue color of
on the reddish brown zone, three strains were the dye was decolorized to colorless. The selected
designated as ST01, ST02 and ST03 (Fig. 1). Among strain ST02 decolorized both the dyes effectively.
the three isolated positive organisms, hypersecretory The pure culture of ST02 on PDA was rich white
strain was selected by quantitative production of aerial mycelium (Figs. 4a & b). The reverse of the
laccase in the potato dextrose broth. Significant mycelia was colorless, pigmentation was not
amount of laccase activities were measured for all the produced even after two weeks of cultivation, hence
three strains. Sample was drawn from the culture for the spores were used for identification. In the
every 24 h to assay the production of laccase. Figure 2 lactophenol cotton blue staining, spores were
cylindrical to subcylindrical, thin-walled, hyaline, and
not amyloid or dextrinoid, without germ pore. The
hyphal system may be monomitic or dimitic,
without binding hyphae. As the morphology was
uninformative and ambiguous, rDNA sequence
comparison was used for identification.
Genomic DNA was successfully isolated from the
selected fungus ST02. The isolated DNA was
confirmed by staining with ethidium bromide viewed
under transilluminator (Fig. 5a). The genomic DNA
was subjected to PCR-amplification of the ITS region

Fig. 1 — Oxidative polymerization of guaiacol to form reddish

brown zones in the medium by positive strains.

Fig. 2 — Laccase production by the selected three positive strains Fig. 3 — Poly-R478 oxidation by ST02 (a) Day 0, (b) Day 6;
in potato dextrose broth. RBBR oxidation by ‘ST02’, (c) Day 0 d) Day 6.
 RATHINASAMY & THAYUMANAVAN: DYE DECOLORIZING LACCASE PRODUCING FUNGI FROM KOLLI HILLS 37

of the rDNA using ITS1 and ITS4 primers. The 5.8S rDNA region from ST02 was used. The
approximate number of the base pair of the amplified sequence for the same have been deposited in
PCR products was (~625 bp) determined (Fig. 5b). the GenBank under the following accession
To identify the strain ST02, rDNA sequence number GU967699.1. The phenogram reflecting
comparison was used. The rDNA sequence of ST02 the phylogenetic relationship between ST02 was
was compared with the rDNA sequence of other fungi constructed using data from the BLAST analysis of
present in the database. The obtained closely related the rDNA region (Figs. 6 & 7). The details of the
sequence in the FASTA format and their length varies organisms, respective accession number of sequences
from 763 bp to 605 bp. The collected sequences used to construct the trees are given in the Table 1.
were aligned by clustalW2 (www.ebi.ac.uk/Tools/
clustalW2/index.html.). To confirm the identification, Discussion
Among the 25 samples collected, three strains
showed reddish brown halo around the growing
mycelium in PDA amended with guaiacol, the
substrate for laccase identification. The chromogen
guaiacol is a very sensitive substrate that allows rapid
screening of fungal strains producing extracellular
guaiacol oxidizing enzymes by means of a color
reaction33. The selected three strains produced the
laccase, which catalyzes the oxidative polymerization
of guaiacol to form reddish brown zones in the
medium (Fig. 1). The obtained results have good
agreement with the report of Kiiskinen et al22 for the
isolation of laccase producing fungus. Fungal strains
that show positive reaction in the plate test for the
secretion of laccase were further analyzed in liquid
culture for the quantitative estimation. Among the
three strains ST02 secretes laccase on second day of
the culture whereas, other two strains secrete on third
day. Maximum secretion of laccase was seen on
12th day of the culture (Fig. 2). Among the three
strains, ST02 produced the maximum laccase activity
Fig. 4 — Pure culture of the isolated strain ‘ST02’ (a) and its (410 ± 1.6 U/l). The production of laccase peaks
mycelium structure at 40X (b). at the late stage of cultivation. The above observation

Fig. 5 —(a) Lane 1: Genomic DNA of ST02, Lane 2: DNA Ladder; (b) Lane 1: 5.8S rDNA (625 bp) Lane 2: DNA Ladder.
38 INDIAN J BIOTECHNOL, JANUARY 2018

Fig. 6 — Phenogram constructed from 5.8S rDNA sequence homologous of ST02 (GU967699.1) by neighbor-joining (NJ) method.

shows that the secreted enzyme is not a primary proteolytic activity in submerged fermentation (SmF)
metabolite and it was a secondary metabolite34. On presumably caused subsequent laccase degradation,
15th day onwards the secretion of laccase was which lowered the ultimate laccase production in SmF
decreased in all the three strains. Mazumder et al35 compared to SSF. The above statement correlated
observed that Pleurotus ostreatus have strong with the report of Pant and Adholeya36.
 RATHINASAMY & THAYUMANAVAN: DYE DECOLORIZING LACCASE PRODUCING FUNGI FROM KOLLI HILLS 39

Fig. 7 — Phenogram constructed from 5.8S rDNA sequence of ST02 (GU967699.1) by UPGMA method.

The selected hypersecretory positive strain (ST02) According to Pant and Adholeya20 a strain that turns
was further investigated to analyze the potential the poly R-478 into yellow or colorless was
of decolorization of dyes like Poly R-478 and considered as a ligninolytic enzyme producing
RBBR individually. Dye decolorization and halo organism. The studies of Palmieri et al38 and Erkurt
formation as a result of oxidation of indicator et al39 proved that the decolorization of anthraquinonic
colored compounds were due to lignolytic enzymes dye RBBR was solely due to the laccase activity. The
production22,37. The isolated strain ST02 decolorizes decolorization of RBBR dye was used as a tool
both the indicator dyes successfully (Fig. 3 a, b, c & d). for biodegradation studies it offers an additional
40 INDIAN J BIOTECHNOL, JANUARY 2018

Table 1 — List of sequences producing significant alignments with Pleurotus ostreatus isolate RP7 (ST02) 5.8S rDNA
Accession No Name of the strain Length Q.C* M.I **
GU967699.1 Pleurotus ostreatus isolate RP7 625 100 100
AB286167.1 Pleurotus eryngii 660 86 91
AY265815.1 Pleurotus columbinus strain CBS 281.32 639 86 91
AY265829.1 Pleurotus ostreatus f. florida strain ATCC 38539 638 86 90
AY265829.1 Pleurotus ostreatus f. florida strain ATCC 38539 638 86 90
AY265831.1 Pleurotus ostreatus, f. florida strain ASI 2016 638 86 90
AY265832.1 Pleurotus floridanus strain CCRC 36038 640 86 91
AY265839.1 Pleurotus ostreatus strain CCRC 36249 640 96 91
AY265848.1 Pleurotus spodoleucus strain ASI 2012 640 86 91
AY368663.1 Pleurotus floridanus strain CCRC 36210 640 86 91
AY368665.1 Pleurotus ostreatus strain ASI 2029 640 86 91
AY540325.1 Pleurotus ostreatus strain S042 673 86 91
AY540326.1 Pleurotus sapidus strain S046 669 86 91
AY540327.1 Pleurotus sapidus strain S047 703 86 91
AY540332.1 Pleurotus ostreatus strain S474 678 86 91
AY581431.1 Pleurotus nebrodensis isolate W6 620 84 91
AY581432.1 Pleurotus nebrodensis isolate W7 605 84 91
AY581433.1 Pleurotus nebrodensis isolate W8 624 86 91
AY581434.1 Pleurotus nebrodensis isolate WW 678 86 91
AY854077.1 Pleurotus ostreatus isolate AFTOL-ID 564 630 86 91
DQ077884.1 Pleurotus ostreatus 640 86 91
DQ077886.1 Pleurotus spodoleucus 639 86 91
DQ077887.1 Pleurotus nebrodensis 639 86 91
DQ333236.1 Pleurotus eryngii clone 3 692 86 91
EF458642.1 Pleurotus ostreatus strain MUCL 28511 763 90 90
EF458649.1 Pleurotus sajor-caju strain MUCL 31674 710 86 91
EF514247.1 Pleurotus ostreatus strain CGMCC 5.37 629 86 91
EF514248.1 Pleurotus ostreatus voucher HMAS 66080 662 86 91
EU424284.2 Pleurotus cornucopiae strain ACCC50234 617 82 92
EU424300.2 Pleurotus ostreatus strain CBS 593.82 679 86 91
EU424301.2 Pleurotus cornucopiae strain ACCC50375 630 83 92
EU424316.2 Pleurotus sapidus strain ACCC50155 679 86 91
FJ501549.1 Pleurotus ostreatus/Coprinus comatus fusant isolate SMCC4.11.15 661 86 91
FJ501564.1 Pleurotus ostreatus isolate SMCC4.03.14 661 86 91
FJ501571.1 Pleurotus ostreatus/Coprinus comatus fusant isolate SMCC4.11.7 660 86 91
FJ810181.1 Pleurotus sapidus strain dd08093 703 86 91
Q.C* = Query Coverage M.I** = Max Identity

advantage compared to usual substrates. Moreover, simple method for screening the bioprospecting fungi
dyes are stable, soluble, possess high molar extinction with lignolytic enzymes for industrial applications.
coefficients and low toxicity and can be applied in The morphological characters of the isolated strain
simple, rapid and quantitative spectrophotometric spores were uninformative and confused with other
assays. RBBR has widely been used since this related genera (Fig. 4a & b). Hence, the molecular
compound represents an important group of method was used for the identification of isolated
organopollutants40-41. The correlation between the strain. The ITS regions of fungal rDNA have been
guaiacol and polymeric dyes were good. The color successfully used for species identification20. The
reactions with synthetic dyes and guaiacol are more isolated strain ST02 was identified using rDNA
easily detectable, detect more laccase positives, and homology. Figures 5a & b shows the isolation and
these compounds can thus reliably be used for laccase amplification of 5.8S rDNA of isolate ST02 from the
activity screening22. According to Masalu42 the plate- genomic DNA, which was ~625 bp, this was closer to
test with indicator compounds is an efficient and other similar sequences reported in NCBI database in
 RATHINASAMY & THAYUMANAVAN: DYE DECOLORIZING LACCASE PRODUCING FUNGI FROM KOLLI HILLS 41

Table 1 (AY265829.1; AY854077.1; EF514247.1). coverage 90% maximum identity to the isolated
The 5.8S rDNA sequence of ST02 (GU 967699.1) selected strain. Zervakis et al48 used the 5.8S rDNA
was blasted in the nucleotide sequence data base of regions as nucleotide sequence divergence portion
PubMed and the first blast hit was Pleurotus ostreatus for molecular phylogeny of mushroom species
strain MUCL 28511 (EF458642.1) which showed Pleurotus cystidiosus and allied taxa. The above
90% query coverage and maximum identity. discussion gives strong correlation with 5.8S rDNA
Another organism P. ostreatus strain CCRC 36249 sequence with UPGMA phyogenetic analysis. The
(AY265839.1) shared maximum query coverage of above organism was deposited and acknowledged by
96% with the ITS sequence of the isolated strain Commonwealth Agricultural Bureaux International
ST02. Fungi showing more than 90% similarity in the (CABI) as “Pleurotus ostreatus IMI 395545”.
5.8S ITS sequence can be considered as belonging to
the same species43, whereas for 18S rDNA sequence Conclusion
homology needs  99% sequence identity can safely This study showed that high laccase producers can
be considered as belonging to the same genus44. Since be discovered from environmental samples by very
the 5.8S rDNA is a hyper variable region, the simple plate test screening methods. Guaiacol is a
evolutionary distance resolved by ITS is usually sensitive substrate for screening the laccase producing
restricted to demarcating within the species level organisms. Dye decolorization potential of the fungus
and cannot be completely relied. The 5.8S rDNA was also determined for the future applications. Using
sequence homology leads to the conclusion that the 5.8S rDNA sequences, a number of fungi species
isolated strain ST02 (GU 967699.1) belongs to the have been identified. The present study has proved
genus Pleurotus and species ostreatus. Peterson et al45 that 5.8S rDNA sequence analysis is a valuable tool
reported three Penicillium sp based on the ITS for the detection of genus and species of unknown
sequence and large subunit rDNA. Following a fungus. In addition, their evolutionary relationships
similar approach, a new fungal strain of Bjerkandera could provide an important clue for further
audusta was identified by Kowalska et al46. exploration of new strains. NJ and UPGMA method
of phylogenetic analysis is more reliable method to
A few other facts also correlated with the find new fungal strains based on ITS sequence.
identification of the strain ST02. Phylogenetic tree
was constructed by two different methods (Neighbor Reference
joining and UPGMA method) for the obtained 1 Kunamneni A, Camarero S, Garcia-Burgos C, Plou F J,
related ITS sequence of the isolated strain. The main Ballesteros A et al, Engineering and applications of
fungal laccases for organic synthesis, Microb Cell Fact,
virtue of NJ relative to these other methods is 7 (2008) 32.
its computational efficiency. Unlike the UPGMA 2 Bustamante M, Gonzalez M E, Cartes A & Diez M C,
algorithm for phylogenetic tree reconstruction, NJ Effect of soya lecithin on the enzymatic system of the
does not assume that all lineages evolve at the same white-rot fungi Anthracophyllum discolour, J Ind Microbiol
Biotechnol, 38 (2010) 189-197.
rate (molecular clock hypothesis) and produces an 3 Eugenio M E, Carbajo J M, Martin J A, Gonzalez A E &
unrooted tree. Phenogram was constructed using NJ Villar J C, Laccase production by Pycnoporus sanguineus
method and the evolutionary distance calculated by under different culture conditions, J Basic Microbiol,
the Tamura-Nei model shows that the strain 49 (2009) 433-440.
4 Sadhasivam S, Savitha S, Swaminathan K & Lin F H,
P. ostreatus voucher HMAS 66080 (EF514248.1)
Production, purification and characterization of mid-redox
was very close to the isolated strain (Fig. 6). Alam et potential laccase from a newly isolated Trichoderma
al47 reported that the size of ITS1 and ITS2 region harzianum WL1, Process Biochem, 43 (2008) 736-742.
varied among the strains and ITS2 was more variable 5 Monteiro M C & De Carvalho M E A, Pulp bleaching using
than that of ITS1, whereas the 5.8S sequence was laccase from Trametes versicolor under high temperature
and alkaline conditions, Appl Biochem Biotech, 70 (1998)
identical to the Pleurotus sp. Figure 7 shows the 983-993.
phenogram, constructed by UPGMA method with 6 Nishida T, Yoshinori K, Mimura A & Takahara Y, Lignin
the same sequence that was used for NJ method and biodegradation by wood-rotting fungi I. Screening of lignin
the distance was calculated by Tamura-Nei model, degrading fungi, Mokuzai gakkaishi, 34 (1988) 530-536.
7 De Jong E, De Vries F P, Field J A, van der Zwan R P &
P. ostreatus strain MUCL 28511 (EF458642.1) was De Bont J A M, Isolation and screening of basidiomycetes
very close to the isolated strain ST02, moreover this with high peroxidative activity, Mycological Res, 12 (1992)
was only strain which shares 90% of and query 1098-1104.
42 INDIAN J BIOTECHNOL, JANUARY 2018

8 Barbosa A M, Dekker R F H & St Hardy G E, Veratryl 25 Tien M & Kirk T K, Lignin peroxidase of Phaneorchaete
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