Journal of Scientific & Industrial Research
Vol. 73, May 2014, pp. 331-337
Strain improvement of white rot fungi Pycnoporus cinnabarinus with the
influence of physical and chemical mutagens for enhancing Laccases production
Rasheeda Khanam*1 and R. Gyana Prasuna2
1
Department of Microbiology, A. Q. J. degree & P.G. College, Visakhapatnam
Department of Microbiology, GITAM Institute of science, GITAM University, Visakhapatnam
2
Received 23 July 2012; revised 6 October 2013; accepted 31 January 2014
Several microorganisms including fungi and bacteria produce the most important industrially applicable enzyme
“laccases”. The present work was aimed to apply mutagenesis on the test fungus Pycnoporus cinnabarinus for enhancement
of the enzyme production. The efficiency of Laccases production by the wild fungal strains Pycnoporus cinnabarinus was
investigated by the treatment with physical mutagen [ultraviolet radiation (UV) and X-rays] and chemical mutagens
[Ethidium bromide, Colchicine and Hydrogen peroxide]. The effect of X-rays showed an increase in production with
increasing exposure (Max. at 8 sec.). Beyond 8 seconds there was a decrease in production. UV irradiation influenced by
reducing the enzyme production and the maximum dosage is lethal to the fungus. Among the three chemical mutagens,
hydrogen peroxide was found to be having lethal effects to the fungus and low enzyme production even in minimum
concentrations. Colchicine and Ethidium bromide showed increase in enzyme production with increasing concentrations
(Max. at 4 and 7 μg respectively). The improved strain of Pycnoporus cinnabarinus showed 15% of increase in the yield.
The increase in production of laccases in a cheap production medium formulated by using agricultural and industrial wastes
may be beneficial industrially when compared to the other costly conventional media. Further work is in progress by
protoplast fusion of the best mutants for even more production.
Key words: P.cinnabarinus, UV rays, X-rays, Ethidium bromide, Colchicine, Hydrogen peroxide, Laccases.
Introduction
Several microorganisms produce many types of
enzymes among which laccases are one of the most
industrially important enzymes. Laccases are copper
containing 1, 4-benzenediol: oxygen oxidoreductases
(EC 1.10.3.2). These are glycosylated polyphenol
oxidases containing 4 copper ions per molecule that
carry out oxidation of phenolic and its related
compound and reduce oxygen to water1, 2.
Laccases has various industrial applications3 such
as textile dye bleaching, pulp bleaching and
bioremediation. In order to improve the color and
quality of the Kraft pulp, chlorine based bleaching
is adopted followed by discharging of waste
waters containing chlorinated aromatics into water
bodies4.Water from such contaminated sources have
cytotoxic and cytomutagenic effects on various living
organisms ultimately harming human beings too. A
similar process is also observed in textile industries
using harmful cytotoxic coloring dyes as the effluents
are released in water bodies5. Fungal laccases are
——————
*
Author for correspondence
Email: [email protected]
environment friendly and help us reduce pollution and
the toxicity of the currently used chemicals through
their oxidation/reduction mode of action6.
A mutagen is a chemical or physical agent that
causes mutations. UV radiation of 260 nm induces
dimerization of adjacent pyrimidine bases, especially
if these both are thymines, results a cyclobutyl dimer.
UV-induced dimerization usually results in a deletion
mutation when the modified strand is copied. Another
type of UV-induced photoproduct is the (6-4) lesion
in which carbons number 4 and 6 of adjacent
pyrimidines become covalently linked7.
Ionizing radiation has various effects on DNA
depending on the type of radiation and its intensity.
Point, insertion and/or deletion mutations might arise,
as well as more severe forms of DNA damage that
prevent subsequent replication of the genome. X-rays
are the ionizing radiation that acts directly on DNA.
Mostly damage is caused indirectly when molecules
around the DNA such as water, are ionized, creating
free-radicals, substances with unpaired electrons.
Most often, the result is single or double stranded
breaks in the DNA molecules. These breaks are hard
to repair because they often leave a phosphate tacked
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J SCI IND RES VOL 73 MAY 2014
onto the 3' OH, where the break occurs7.
Colchicine is an effective chemical mutagen. It
binds to tubulin and prevents its polymerization into
microtubules8.
Ethidium bromide is one of the best known
intercalator. It is a positively charged polycyclic
aromatic compound which binds to DNA by inserting
itself between the base pairs by a process called
intercalation. Its binding to DNA causes a local
unwinding of the helix by around 260 and alteration in
the shape of the molecule7.
Hydrogen peroxide (H2O2) can induce a variety of
genetic alterations, probably by the generation of
hydroxyl radicals via the Fenton reaction9.
With an aim of improving the strain of white rot
fungus by the above said mode of action of the
respective mutagens the present work was carried out
by applying mutagenesis by physical and chemical
mutagens to enhance the production of laccases
quantitatively.
White rot fungi P.cinnabarinus was maintained as
a pure culture in PDA plates and PD broth medium.
LAMP (VL-G), UVtube T-15C 15W 254 nm,
VILBER-LOURMAT) where the distance between
the agar surface and the lamp was adjusted to 30 cm.
A non-irradiated plate was maintained as control.
After irradiation the mycelial suspensions were
incubated at 300C overnight in dark10. Later the plates
were incubated at 300C in an incubator for 7days until
the fungal colonies were observed.
A similar procedure was followed to treat the
parent strains with X-rays. The freshly prepared
screening medium plates were exposed to X-rays for
the time intervals-2sec, 4sec, 6sec, 8sec, 10sec, 12sec,
14sec, 16sec, 18sec and 20sec under GEDX 300mg at
52-54mA. A non-irradiated plate was maintained as
control. After irradiation the mycelial suspensions
were incubated at 300C overnight in dark. Later the
plates were incubated at 300C in an incubator for
7days until the fungal colonies were observed. The
surviving colonies were examined for some characters
including growth rate that measured as linear growth
(cm), colony morphology for any change in the color
or texture and quantity of enzyme production. The kill
curve was prepared and time of exposure was
optimized for the mutation of P.cinnabarinus for
hyper production of laccases.
Mutagenesis
Mutagenesis by chemical mutagens
Materials and methods
Microorganisms
Preparation of agar plates
Screening agar medium was prepared as per the
composition. 3.0 peptone, 10.0 glucose, 0.6 KH2PO4,
0.001 ZnSO4, 0.4 K2HPO4 0.0005 FeSO4, 0.05
MnSO4, 0.5MgSO4, 20.0 agar (pH-6) supplemented
with 0.2% Guaiacol. The medium was poured in
molten condition into the petriplates and allowed to
solidify.
Inoculation of the test fungi
The test fungus was inoculated in the well labeled
petriplates. The labeling was done according to the
type of mutagen, concentration or the time of
exposure.
Mutagenesis by UV rays and X-rays
The mutants were obtained from P.cinnabarinus by
treating with UV-rays and X-rays. The parent strain of
P.cinnabarinus was grown on PDA slants at 30oC for
1week. After one week the white mat of fungal
growth was suspended from the agar surface to a
freshly prepared screening medium plates and was
irradiated for different time intervals-2mins, 4mins,
6mins, 8mins, 10mins, 15mins, 20mins, 25mins and
30mins under ultraviolet lamp (GERMICIDAL
A stock of 10 micrograms of the chemical mutagen
per ml was prepared. A series of concentrations 1 -10
micrograms were prepared from the stock. The test
fungus grown in PDA medium at 300C for one week
was suspended in a series of sterile screening medium
plates containing varied concentrations 1-10
micrograms of either mutagen (colchicine, hydrogen
peroxide or ethidium bromide). All the plates were
appropriately labeled with the type of mutagen and its
concentration. The plates were then incubated at 300C
overnight in dark. Simultaneously a control was
maintained without any chemical mutagen for
comparison. Later the plates were shifted in an
incubator at 300C for 7 days. The surviving colonies
were examined for some characters including growth
rate that measured as linear growth (cm), colony
morphology for any change in the color or texture and
quantity of enzyme production.
Selection of mutants
Following steps were adopted in order to select the
specific mutant having the ability to hyper produce
laccases.
*Selection based on colony formation
KHANAM & PRASUNA: STRAIN IMPROVEMENT OF WHITE ROT FUNGI
*Selective marker
*Enzyme diffusion zone test
*Quantification of enzyme
Selection based on colony formation
After treating the fungal cultures with above five
mutagens the surviving fungus forming colony were
selected and sub cultured thrice in the screening
medium in order to observe its survival and its
laccases production property.
Isolation of mutants using Selective marker
The surviving colonies were inoculated in a
screening medium in which Guaiacol was used as a
selective marker and the mutants were selected based
on the intensity of color produced on screening
medium plates after 7 days of incubation at 300C.
333
from four different points and the average radius was
recorded. Measurements were taken at 24 hours
intervals over a period of 6days. A light fitted to the
colony counter with a magnifying glass enabled
accurate measurement of the fungal growth.
The average rate of mycelial growth (cm day-1) was
determined over a four day period. To determine this,
the following calculation was employed:
[G (d6-G (d5)] + [G (d5) – G (d4)] + [G (d4) – G
(d3)] + [G (d3) – G (d2)]} ÷ 4
Where: G (d6) is equal to the average mycelial growth
(in cm) on day 6 and G (d5) is equal to the average
mycelial growth (in mm) on day 5. If the mycelial
growth had reached the perimeter of the plate before
day 6, then day 5 was used as the starting point for the
calculation.
Qualitative screening of mutants/enzyme diffusion zone test
The screening medium was prepared by adding all
constituents and then autoclaved and dispensed into
sterile petridishes. The plates were inoculated with the
surviving colonies and incubated at 30oC for a period
of 5-7 days. The development of dark reddish brown
zone is an indication of laccases activity, and its area
is a measure of the extent of activity. At regular
intervals of 24 h incubation, each plate was examined
and measurements on the area of the colored zone
were taken to monitor laccases activity 11. The strain
showing the greatest diffusion areas (mm) were
further studied.
Quantification of enzyme
All the surviving colonies after treating with five
mutagens were inoculated in the best laccases
production medium (PD WBG) and incubated in an
orbital shaking incubator at 120 rpm at 28-300C for
the optimum time period12. After incubation the
enzyme produced by the mutants was assayed
spectrophotometrically.
Estimation of growth rate of wild and mutant
Radial growth measurements were performed
following the method of Lonergan et al. (1993). The
radial zone measurements were performed in
triplicate on the fungal colonies grown on PDA. PDA
was used in preference to MEA because of its
translucent nature, which enabled the growing edge to
be seen and measured clearly.
Growth assays were performed on all the plates by
measuring the mycelial radius (minus the plug radius)
of the colony in cm. The measurements were taken
Molecular sequencing
DNA was isolated from the culture sample. Its
quality was evaluated on 1.2% Agarose Gel.
Fragment of D1/D2 region of LSU (Large subunit
28S rDNA) gene was amplified by PCR from the
above isolated plasmid DNA. The PCR amplicon was
purified to remove contaminants. Forward and reverse
DNA sequencing reaction of PCR amplicon was
carried out with DF and DR primers using BDT v3.1
Cycle sequencing kit on ABI 3730xl Genetic
Analyzer.
Consensus sequence of 655 bp of D2 region of 28S
rDNA gene was generated from forward and reverse
sequence data using aligner software. The D1/D2
region of LSU (Large subunit 28S rDNA) gene
sequence was used to carry out BLAST with the
nrdatabase of NCBI GenBank database. Based on
maximum identity score first ten sequences were
selected and the Phylogenetic tree was constructed
using MEGA 4 13.
Results and discussion
Mutants of P.cinnabarinus were obtained by
treating it with physical and chemical mutagens with
a view to enhance the laccases enzyme production.
Effect on growth
The physical mutagen X-rays caused a decrease in
the growth rate of the fungal colonies (growth
measured in diameter of the colony in cm) with
increasing time of exposure and was almost lethal to
the fungi at maximum time of exposure (20 sec).
Even if any live colony was found at this maximum
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time of exposure, its laccases producing property was
completely lost.
The fungal growth rate was proportionately
decreasing with increasing the time of UV rays
exposure till 30 minutes. But it was also found that
the growth rate was not constantly decreasing after 30
minutes of UV exposure. The effect of chemical
mutagens was more on its growth rate when
compared to the physical mutagens. The diameter of
the colony was decreasing with increasing
concentration of the chemical mutagen. The effect of
Hydrogen peroxide was lethal at higher concentration.
Thus hydrogen peroxide is more effective to the
fungal culture among all the three mutagens
(colchicine, ethidium bromide and hydrogen
peroxide).
Screening of mutants based on enzyme diffusion zone
The diameter of the colored zone of Guaiacol
oxidation, denoting laccases activity on the screening
medium plate, after 7 days of incubation was
maximum with the strain treated for 8 seconds (KX8)
(4.0±0.1cm) with X-rays when compared to the wild
one (KR) (3.2±0.1cm). On the other hand the effect of
UV exposure resulted in reduction of laccases
activity. All the living strains obtained after UV
exposure were not producing more zone of Guaiacol
oxidation than the wild one. Thus UV exposure was
not used any further for obtaining mutations
(Table.1).
Among the three chemical mutagens, the effect
of colchicines with 4µg/ml concentration showed
maximum zone of enzyme diffusion (3.6±0.3cm).
Next to this concentration 3µg/ml and 7µg/ml
concentrations were showing the zone of oxidation
more than the wild one. Neither ethidium bromide nor
hydrogen peroxide was found to have positive effect
on laccase production (Table.2)
Isolation of mutants
Quantification of enzyme
of
P.cinnabarinus
based
on
With respect to the quantitative estimation of the
enzyme produced by the mutants, there was a
maximum enzyme production by a mutant (KX8)
obtained by X- ray exposure for 8 minutes. It resulted
in 12.9% more productivity when compared to the wild
one (KR) (104.52U/ml/min), where as the strain
improvement was not observed in the strains after
exposure to UV rays. Neither of the strains obtained
after UV treatment were able to produce laccases in
higher quantity than the wild one. Similar to the
observations earlier among the three chemical
mutagens colchicine was found to be having more
positive effect on strain improvement with 4µg per ml
concentration. The strain obtained by treating with 4µg
per ml of colchicine could produce 11.4% more
laccases quantitatively. Ethidium bromide showed
decreased laccases production at all concentrations
except at 7 µg per ml concentration resulting 6.6%
increase. As observed earlier hydrogen peroxide caused
reduction in enzyme production and was also lethal to
the fungus at higher concentrations (Graph.1).
The efficiency of laccases production by
P.cinnabarinus was thus increased by approximately
Table 1—Diameter of enzyme diffusion zone after treating with physical mutagens -P.cinnabarinus
X-rays
UV rays
Time
Diameter of zone (cm)
(Second)
UV rays
Time (Minutes)
diameter of zone (cm)
Time
diameter of zone
(Minutes) (cm)
0
3.2± 0.1
0
3.2± 0.2
70
2.6± 0.3
2
3.0 ± 0.2
2
2.4 ± 0.3
80
3.2± 0.3
4
3.2 ±0.1
4
2.4 ± 0.1
90
3.2± 0.2
6
2.8 ± 0.3
6
2.6 ± 0.2
100
2.6± 0.1
8
4.0 ± 0.1
8
2.8± 0.3
110
2.8± 0.1
10
3.6 ± 0.3
10
2.8 ± 0.2
120
2.8± 0.1
12
2.8 ± 0.3
20
3.0± 0.2
14
2.8 ± 0.2
30
3.0 ± 0.1
16
3.0 ± 0.2
40
3.0 ± 0.2
18
3.0 ± 0.1
50
3.0 ± 0.1
20
0
60
2.2 ± 0.2
Comparison of wild and the mutant strains of P.cinnabarinus obtained after X-rays and UV rays based on the zone of enzyme diffusion
(cm) on screening medium. Each value is the average of triplicate. ± indicates standard deviation among replicates. The mutants after
treatment with X-rays were named as KX2, KX4, KX6….. and KX20.The mutants after treatment with UVrays were named as KUV2,
KUV 4, KUV 6….. and KUV120.
KHANAM & PRASUNA: STRAIN IMPROVEMENT OF WHITE ROT FUNGI
335
Table 2—Diameter of enzyme diffusion zone after treating with
chemical mutagens P.cinnabarinus
(μg/ml)
0
1
2
3
4
5
6
7
8
9
10
Colchicine
3.2± 0.2
2.8 ± 0.1
2.8 ± 0.3
3.4 ± 0.2
3.6 ± 0.3
3.0 ± 0.1
3.2 ± 0.2
3.4 ± 0.2
2.8 ± 0.3
2.8 ± 0.2
2.6 ± 0.2
Ethidium
bromide
3.2± 0.2
2.0 ± 0.1
2.2 ± 0.3
2.6± 0.2
3.0± 0.1
2.4 ± 0.2
2.4 ± 0.2
3.4 ± 0.3
3.0 ± 0.2
2.6 ± 0.1
2.4 ± 0.2
Hydrogen
peroxide
3.2± 0.2
2.8± 0.1
2.8± 0.1
2.0± 0.3
2.2± 0.2
3.0± 0.2
3.0± 0.1
2.0± 0.3
1.2± 0.3
0
0
Comparison of wild and the mutant strains of P.cinnabarinus
obtained after X-rays and UV rays based on the zone of enzyme
diffusion (cm) on screening medium. Each value is the average of
triplicate. ± indicates standard deviation among replicates.The
mutants after treatment with Colchicine were named as KC1, KC2,
KC3…… and KC10. The mutants after treatment with Ethidium
bromide were named as KE1, KE2, KE3…… and KE10. The
mutants after treatment with Hydrogen peroxide were named as
KH1, KH2, KH3…… and KH10
Graph 1—Effect of chemical mutagens on Laccases production
Fig. 1—Morphological difference between the wild and mutant fungus a, b=Wild form of P.cinnabarinus(KR); c, d=Mutant form
P.cinnabarinus(KX8)
13% when treated with physical mutagen X-rays.
Though the effect of chemical mutagens like colchicine
and ethidium bromide was also proving to increase its
productivity, the effect of X-rays at 8 seconds
of exposure was showing maximum variation when
compared to the others. Thus, the mutant strain of
P.cinnabarinus obtained by treating with X-rays for
8 seconds, exhibiting maximum laccases production
among all other strains was named as KX8 and
later KM for convenience and used for further studies.
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J SCI IND RES VOL 73 MAY 2014
Fig. 2—Sequence producing significant alignments
Morphological differences
The morphological appearance of the fungal strain
treated with x-rays for 8 seconds showed remarkable
differences when compared with the wild one when
grown on PDA medium plates for one week.
The wild form of P.cinnabarinus colony was purely
white, cottony and fuzzy but the mutant was appearing
yellowish, cottony, thicker and less fuzzy (Fig.1).
Improvement in growth rate
The growth rate of mutant was observed to be
greater than the wild one. The growth rate of the
mutant (KX8) was 2.15/day which was about 13%
more than the growth rate of the wild one (1.9/ day).
Differences at molecular level
The difference between the wild (KR) and mutant
(KX8) at molecular level was done by 28S rDNA
sequencing. A single band of high-molecular weight
DNA has been observed on 1.2% agarose gel. A
single discrete PCR amplicon band of 650 bp was
observed when resolved on agarose Gel.
The DNA sequence analysis of the mutant strain of
P.cinnabarinus was performed which was granted
with an Accession number 56259 by NCBI BLAST. A
few base pairs were different in the mutant strain
when compared to the wild strain. The differing base
pairs were shown in red (Fig.2). The nitrogen base
KHANAM & PRASUNA: STRAIN IMPROVEMENT OF WHITE ROT FUNGI
pairs of wild strain (KR) positioning at 113, 115, 116,
226, 245, 557, 558, 559, 570, 571, 574 were
mismatched with the mutant one (KX8). Transition
type of base substituting point mutation was observed
at positions 113, 115, 116, 574, 557, 558, 559 and
transversion type of point mutation was observed at
226, 245, 570 and 571. There were two gaps formed
between the base pairs 567 and 568 in the wild culture
sequence due to insertion mutation observed in the
mutant strain. The further studies on characterization,
purification and even more development of the strain
by protoplast fusion were carried out with this mutant
of P.cinnabarinus (KX8).
Acknowledgement
The authors are thankful to Prof M. A. Singara
charya, Department of Microbiology, Kakatiya
University for providing the fungal cultures and a
moral support during the tenure of the work.
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