Abstract: 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.
INTRODUCTION
Laccase (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) 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 the species.
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 enzymes.
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 as standard.
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).
| Table 1: | Production of laccase (±SE) in different Pleurotus spp. in PD medium using 25th day’s culture filtrate |
| 1 Specific activity was measured in terms of o-dianisidine oxidizing activity | |
| Fig. 1: | 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. 1).
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, Fig. 2).
| Fig. 2: | Dry weight of 25 day’s grown mycelium of different Pleurotus spp. The bar showed the standard error |
| Fig. 3: | Zymogram of different Pleurotus species (developed by o-dianisidine) from 25th day’s 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.
ACKNOWLEDGMENTS
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.