Experimental Evidence for Presence of a Growth Regulating Extracellular Laccase in Some Pleurotus Species
The aim of the present study was to find out a common laccase isoenzyme in five Pleurotus spp. which might be responsible for regulation of mycelial growth in producer organisms. Laccase activities were assayed from the extracellular culture filtrates and optimum dates for laccase production under stationary condition were detected. Partially purified 25th days culture filtrates were analyzed for activity staining in 10% Native PAGE. The culture filtrate of all the strains had shown the oxidization of o-dianisidine, guaiacol and ABTS with different efficiencies. P. ostreatus showed optimum laccase activity in 25th day whereas other strains shown optimum activities in 26th day. The highest laccase activity and biomass were found in P. ostreatus and lowest in P. florida. Though different Pleurotus species exhibited differential laccase isoenzyme expression patterns but one isoenzyme band had been found to be common in all the species, which resulted in similar rf value with that of a previously reported growth regulating laccase (L2) of P. florida.
to cite this article:
N. Das, S. Naskar, P. Chowdhury, B. Pasman and D. Adhikari, 2011. Experimental Evidence for Presence of a Growth Regulating Extracellular Laccase in Some Pleurotus Species. Research Journal of Microbiology, 6: 496-502.
Received: February 28, 2011;
Accepted: April 14, 2011;
Published: May 21, 2011
Laccase (benzenediol: oxygen oxidoreductase; EC 220.127.116.11) is a common extracellular
copper containing enzyme in white rot fungi including Pleurotus sp. (Mayer,
1987; Youn et al., 1995; Khammuang
and Sarnthima, 2007; Mukherjee and Das, 2009). Different
isoenzymes of laccase have been the results of extracellular secretions in fungal
species depending on the environmental conditions (Giardina
et al., 1999). Apart from its main role in lignin degradation (Galliano
et al., 1991; Thurston, 1994) various other
functions of laccase has been suggested by investigators viz. rapid cell growth
and formation of primordia (De Vries et al., 1986),
detoxification of pollutants (Eggert et al., 1997;
Desai and Nityanand, 2011), pathogenesis (Vaiterbo
et al., 1994; Galhaup et al., 2002)
etc. Laccase is very much stable and easy to handle enzyme so utilized in a
number of biotechnological purposes (El -Shora et al.,
2008; Gilaki, 2010).
Oyster mushroom (Pleurotus spp.) enjoys second position after the button
mushroom (Agaricus bisporus) in terms of yield/ production turnover around
globe (Sanchez, 2010). Pleurotus spp. are not
only regarded as edible mushroom but produced a number of pharmaceutically important
substances (Tambekar et al., 2006). The yield
of this very mushroom varies not only from species to species but also in between
strains of the same species. There is hardly any reported marker character of
this mushroom which can be helpful for the assessment of yield before cultivation.
The present investigator has reported that one extracellular laccase enzyme
(L2), produced by P. florida, is associated with the regulation
of mycelial growth, a prerequisite for fruit-body production (Das
et al., 1997, 2001). In the present study,
it is tried to enumerate that P. florida shares the similar laccase isoenzyme
profile with P. ostreatus and irrespective of the differential laccase
isoenzyme patterns in different Pleurotus species, a common growth regulating
laccase-like isoenzyme (L2 of P. florida) is present in all
MATERIALS AND METHODS
Strain and culture media: The mycelial strain of P. florida (ITCC,
3308) was obtained from Society for Rural Industrialization, Ranchi, India.
P. ostreatus (MTCC, 1802), P. flabellatus (MTCC, 1799), P.
sajorcaju (MTCC, 1806) and P. pulmonarius (MTCC, 1805) were obtained
from Microbial Type Culture Collection and Gene Bank, Institute of Microbial
Technology, Chandigarh, India and maintained in Potato Dextrose Agar (PDA) (Das
and Mukherjee, 2007). Mushroom strains were grown in Potato Dextrose (PD))
media (Das et al., 1997) to obtain the extracellular
Inoculum source and culture conditions: An inoculum (6 mm in diameter) was taken from the periphery of colonies growing on PDA for 10 days. The production of laccase was studied in liquid PD medium in stationary conditions at 25+1°C. Generally the culture filtrate was collected after 25 days of growth wherever not mentioned. The culture supernatant was obtained by centrifugation (10000 gx30 min) and used as the source of enzyme.
Partial purification of laccase: The laccase activity present in the culture filtrate was partially purified after 80% ammonium sulphate precipitation, followed by extensive dialysis against 0.01 mM acetate buffer (pH 5.0).
Assay for laccase activity: Laccase activity was assayed spectrophotometrically
as described by Das et al. (1997) with guaiacol
or o-dianisidine as the substrates. The ABTS activity was assayed according
to Bose et al. (2007), Enzyme activity was expressed
in International Unit.
Protein estimation: Protein concentration was determined according to
the method of Bradford (1976) using bovine serum albumin
Activity staining: Activity staining of enzyme after native Poly Acrylamide
Gel Electrophoresis (PAGE) of the 25th days partially purified culture filtrate
was done with solution of o-dianisidine in acetate buffer, pH 5.0 as reported
earlier (Das et al., 1997; Bose
et al., 2007).
Statistical analysis: All experiments were carried out using 9 replicates (3 setsx3 batches) and best values were determined by a linear least-square regression analysis using MINITAB Version 6.
RESULTS AND DISCUSSION
In the present study, all the tested species of Pleurotus i.e., P.
florida (ITCC, 3308), P. ostreatus (MTCC, 1802), P. flabellatus
( MTCC, 1799), P. sajorcaju (MTCC, 1806) and P. pulmonarius (MTCC,
1805) had been found to produce laccase enzyme in PD medium (Table
1). The culture filtrate of all the strains could effectively oxidize o-dianisidine,
guaiacol and ABTS. ABTS oxidizing ability of all the strains was found to be
much higher over the other two substrates (Table 1).
|| Production of laccase (±SE) in different Pleurotus
spp. in PD medium using 25th days culture filtrate
|1 Specific activity was measured in terms of o-dianisidine
||Day wise (14th-28th ) laccase activity of different Pleurotus
species. Enzyme activity was measured by o-dianisidine
The efficiency for extracellular laccase production was found to vary not
only from species to species but also in different cultural condition. Heterogeneous
laccase production patterns had been reported in several times in various fungi
depending on the media compositions and/other influencing conditions (Youn
et al., 1995). Reports are available where P. ostreatus had
been found to be the topper in laccase production among the different members
of oyster mushrooms. Interestingly, in the present study, P. ostreatus
(1802) was able to produce highest quantity of laccase in the 25th day of culture,
whereas the other species could even effectively exhibit their optimum laccase
activities in the 26th day of culture (Fig. 1). P. florida
(3308) had been found to demonstrate the lowest enzyme activity using o-dianisidine
or guaiacol as the substrates (Table 1, Fig.
Though all the strains of Pleurotus were found to produce laccase from
the very early phase of growth (data not shown) but the optimum laccase activity
of different strains could only be observed during 25 to 26 days (Fig.
1). The time period for laccase production also reported to vary in different
species, owing to several influencing factors prevalent in cultural conditions,
like temperature, pH, media compositions, stationary or shaking conditions etc.
Different studies have already reported that ligninolytic enzyme activities
get triggered as a result of depletion of nutritional factors and the notable
enzymes like laccase was found to be produced at the stationary period of growth
phase (Kirk and Farrel, 1987; Higuchi,
1990; Guillen et al., 1992).
In the present study, most of the Pleurotus strains had been showing
a positive correlation between laccase production and mycelial growth as evidenced
by comparative laccase activity and mycelial dry wt. (Table 1,
||Dry weight of 25 days grown mycelium of different Pleurotus
spp. The bar showed the standard error
||Zymogram of different Pleurotus species (developed
by o-dianisidine) from 25th days partially purified culture filtrate.
1-3308, 2-1802, 3-1805, 4-1799, 5-1806
When the specific activities were measured in terms of mycelia or protein,
strain 1802 (P. ostreatus) could exhibit the maximum activity whereas
strain 3308 (P. florida) was the weakest in laccase enzymatic potentials
(Table 1). The mycelial dry weight was also found to be highest
in strain 1802 and lowest in 3308 (Fig. 2). Das
et al. (1997) had propounded a connection on the involvement of a
particular laccase isoenzyme (L2) of P. florida during the
vegetative growth phase and recommended that highly productive strains were
also found to produce large amounts of laccase in to the extra cellular medium.
Mansur et al. (1997) reported that higher amount
of extracellular laccase might help in efficient substrate invasion which could
promote the growth and productivity effectively. Tlecuitl-Beristain
et al. (2008) reported the positive correlation being existed between
laccase activity and growth phase of the fungus Pleurotus ostreatus.
In present study, all the tested strains have shown two or more laccase isoenzymes
(evidenced from non-denaturing zymogram profile) present in extracellular culture
filtrate (Fig. 3). Interestingly, two laccase isoenzymes were
found to be present in strain 3308, 1802 and 1799 while three in strain 1805
and four in 1806 in native PAGE. In the zymogram, a particular laccase band,
which had been found to have similar rf value with L2-laccase
of P. florida (3308) was found to be present prominently in all the lanes(Fig.
3). Different workers reported that most of the fungal members could produce
more than one laccase isoforms although the number might vary from experiment
to experiment (Mayer, 1987; Mukherjee
and Das, 2009; Thurston, 1994). De
Souza et al. (2004) reported at least three laccase isoforms, of
which two could be produced in a non-induced culture condition and one could
be the resultant in a induced culture of P. pulmonarius. Tellez-Tellez
et al. (2005) had reported about two isoforms of intracellular laccase
in two different strains of P. pulmonarius. Stajic
et al. (2006) had reported the presence of three isoenzymes of P.
pulmonarius, whereas Soden and Dobson (2001) could
trace four isoenzymes of laccase in P. sajorcaju. The number of laccase
isoenzymes reported by different workers in P. ostreatus had not been
found to be same. It was reported that six strains of P. ostreatus could
produce two different isoforms of intracelluler laccases. Stajic
et al. (2006) have reported three isoenzymes in P. ostreatus.
Mansur et al. (2003) had reported four laccase
isoenzymes in P. ostreatus but they could locate only two isoenzymes
during guaiacol staining throughout the culture period. Interestingly, during
purification after ultrafiltration and ammonium sulphate precipitation, two
new laccase isoforms were noticed to appear in the native gel, only when ABTS
was used as substrate. Das et al. (1997) reported
two distinct laccase isoforms in P. florida and suggested that within
the two laccase isoforms, the L2-laccase of P. florida was
supposed to be actively responsible for mycelial growth. It has been found that
in native PAGE both P. florida (3308) and P. ostreatus (1802)
have produced two prominent laccase isozyme bands (Fig. 3).
Tellez-Tellez et al. (2005) have suggested that
laccase isoenzyme zymogram profile could be considered as a biochemical parameter
for differentiating different species among the genus Pleurotus. They
had also reported similar laccase isoenzyme patterns in P. ostreatus
and P. florida and been able to comment and highlight on their functional
role in elucidating a probable phylogenetic relationships.
The most important observation in this work is that irrespective of differential laccase activity and isoenzyme patterns, a growth regulating laccase like-enzyme (like L2-laccase of P. florida) has been found to be dominantly present in all the tested Pleurotus spp. as evidenced by their relative movement (rf) in native PAGE (Fig. 3).
From the results, it can be concluded that laccase is an important enzyme in
all the tested strains of Pleurotus sp. The number and position of laccase
band in a species is its unique property. Present observation suggest that irrespective
of divergence in laccase isoenzyme patterns in different Pleurotus spp.,
all of them possessed a unique laccase isoenzyme (as evidenced by zymogram)
which might be correlated to be actively involved in mycelial growth of all
the strains, that could also be a prerequisite for sporophore (fruiting body)
development. So, this particular laccase can be effectively exploited as a marker
enzyme for strain differentiation, improvement and assessment of fruiting efficiency,
prior to commercial cultivation of the oyster mushrooms.
The work was financially supported by Department of Biotechnology, Govt. of India (Sanction order no. BT/PR9769/GBD/27/66/2007). The authors acknowledge the help of Dr. Mina Mukherjee of IICB, Jadavpur, Kolkata.
Bose, S., S. Mazumder and M. Mukherjee, 2007.
Laccase production by the white-rot fungus Termitomyces clypeatus
. J. Basic Microbiol., 47: 127-131.CrossRef | PubMed |
Bradford, M.M., 1976.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254.CrossRef | PubMed | Direct Link |
Das, N., S. Sengupta and M. Mukherjee, 1997.
Importance of laccase in vegetative growth of Pleurotus florida
. Applied Environ. Microbiol., 63: 4120-4122.PubMed | Direct Link |
Das, N., T.K. Chakraborty and M. Mukherjee, 2001.
Purification and characterization of a growth regulating laccase from Pleurotus florida
. J. Basic Microbiol., 5: 261-267.Direct Link |
Das, N. and M. Mukherjee, 2007.
Cultivation of Pleurotus ostreatus
on weed plants. Bioresour. Technol., 98: 2723-2726.CrossRef | Direct Link |
Desai, S.S. and C. Nityanand, 2011.
Microbial laccases and their applications: A review. Asian J. Biotechnol., 3: 98-124.CrossRef | Direct Link |
De Souza, C.G.M., G.K. Tychanowicz, D.F. De Souza and R.M. Peralta, 2004.
Production of laccase isoforms by Pleurotus pulmonarius
in response to presence of phenolic and aromatic compounds. J. Basic Microbiol., 44: 129-136.CrossRef | PubMed | Direct Link |
De Vries, O.M.H., W.H.C.F. Kooistra and K Wessels, 1986.
Formation of an extracellular laccase by a Schizophyllum commune
dikaryon. J. Gen. Microbiol., 132: 2817-2826.CrossRef |
Eggert, C., U. Temp and K.E.L. Eriksson, 1997.
Laccase is essential for lignin degradation by the white-rot fungus Pycnoporus cinnabarinus
. FEBS Lett., 407: 89-92.CrossRef | Direct Link |
El-Shora, H.M., M.M. Youssef and A.S. Khalaf, 2008.
Inducers and inhibitors of laccase from Penicillium
. Biotechnology, 7: 35-42.CrossRef | Direct Link |
Galhaup, C., H. Wagner, B. Hinterstoisser and D. Haltrich, 2002.
Increased production of laccase by the wood-degrading basidiomycete Trametes pubescens
. Enzyme Microb. Technol., 30: 529-536.CrossRef |
Galliano, H., G. Gas, J.L. Sevis and A.M. Boudet, 1991.
Lignin degradation by Rigidoporus lignosus
involves synergistic action of two oxidizing enzymes: Mn peroxidase and laccase. Enzyme Microb. Technol., 13: 478-482.CrossRef |
Giardina, P., G. Palmieri, A. Scaloni, B. Fontanella, V. Faraco, G. Cennamo and G. Sannia, 1999.
Protein and gene structure of a blue laccase from Pleurotus ostreatus
. Biochem. J., 341: 655-663.Direct Link |
Gilaki, M., 2010.
Nano immobilization of enzyme to improvement of biofuel cell electrode's function. Pak. J. Biol. Sci., 13: 611-612.CrossRef | Direct Link |
Guillen, F., A.T. Martinez and M.J. Martinez, 1992.
Substrate specificity and properties of the aryl-alcohol oxidase from the ligninolytic fungus Pleurotus eryngii
. Eur. J. Biochem., 209: 603-611.CrossRef |
Higuchi, T., 1990.
Lignin biochemistry biosynthesis and biodegradation. Wood Sci. Technol., 24: 23-63.CrossRef |
Kirk, T.K. and R.L. Farrell, 1987.
Enzymatic combustion: The microbial degradation of lignin. Annu. Rev. Microbiol., 41: 465-505.CrossRef |
Khammuang, S. and R. Sarnthima, 2007.
Laccase from spent mushroom compost of Lentinus polychrous
Lev. and its potential for remazol brilliant blue R decolourisation. Biotechnology, 6: 408-413.CrossRef | Direct Link |
Mansur, M., M.E. Arias, J.L. Copa-Patino, M. Flardh and A.E. Gonzalez, 2003.
The white-rot fungus Pleurotus ostreatus
secretes laccase isozymes with different substrate specificities. Mycologia, 95: 1013-1020.Direct Link |
Mansur, M., T. Suarez, J.B. Fernandez-Larrea, M.A. Brizuela and A.E. Gonzalez, 1997.
Identification of a laccase gene family in the new lignin-degrading basidiomycete CECT 20197. Applied Environ. Microbiol., 63: 2637-2646.PubMed | Direct Link |
Mayer, A.M., 1987.
Polyphenol oxidases in plants-recent progress. Phytochemistry, 26: 11-20.CrossRef | Direct Link |
Mukherjee, M. and N. Das, 2009.
Fungal Laccase: A Biotechnologically Potential Enzyme. In: Biotechnology Applications, Mishra, C.S.K. (Ed.). I.K. International, New Delhi, ISBN-13: 9789380026299, pp: 70-108
Palmieri, G., P. Giardina, L. Marzullo, B. Desiderio, B. Nitti, R. Cannio and G. Sannia, 1993.
Stability and activity of a phenol oxidase from the ligninolytic fungus Pleurotus ostreatus
. Applied Microbiol. Biotechnol., 39: 632-636.CrossRef |
Sanchez, C., 2010.
Cultivation of Pleurotus ostreatus
and other edible mushrooms. Applied Microbiol. Biotechnol., 85: 1321-1337.CrossRef | PubMed |
Soden, D.M. and A.D. Dobson, 2001.
Differential regulation of laccase gene expression in Pleurotus sajor
-caju. Microbiology, 147: 1755-1763.Direct Link |
Stajic, M., L. Persky, D. Friesem, Y. Hadar, S.P. Wasser, N. Nevo and J. Vukojevic, 2006.
Effect of different carbon and nitrogen sources on laccase and peroxidases production by selected Pleurotus
species. Enzyme Microbiol. Technol., 38: 65-73.CrossRef | Direct Link |
Tambekar, D.H., T.P. Sonar, M.V. Khodke and B.S. Khante, 2006.
The novel antibacterials from two edible mushrooms: Agaricus bisporus
and Pleurotus sajor caju
. Int. J. Pharmacol., 2: 584-587.CrossRef | Direct Link |
Tellez-Tellez, M., C. Sanchez, O. Loera and G. Diaz-Godinez, 2005.
Differential patterns of constitutive intracellular laccases of the vegetative phase of Pleurotus
species. Biotechnol. Lett., 27: 1391-1394.Direct Link |
Thurston, C.F., 1994.
The structure and function of fungal laccases. Microbiology, 140: 19-26.Direct Link |
Tlecuitl-Beristain, S., C. Sanchez, O. Loera, G.D. Robson and G. Diaz-Godinez, 2008.
Laccases of Pleurotus ostreatus
observed at different phases of its growth in Submerged fermentation: Production of a novel laccase isoform. Mycol. Res., 112: 1080-1084.CrossRef |
Vaiterbo, A., B. Yagen and A.M. Mayer, 1994.
Cucurbitacins, attack enzymes and laccase in Botrytis cinerea
. Phytochemistry, 32: 61-65.CrossRef |
Youn, H.D., K.J. Kim, J.S. Maeng, Y.H. Han and I.B. Jeong et al
Single electron transfer by an extracellular laccase from the white rot fungus Pleurotus ostreatus
. Microbiology, 141: 393-398.CrossRef | PubMed |