Effect of metal ions

Effect of metal ions, surfactants and veratryl alcohol on partially purified laccase was evaluated. Metal ions are generally known to alter the enzyme on binding with and cause stabilization and destabilization. Studies performed earlier have shown that several metal ions inhibit the enzyme activity such as by organic solvents, metal ions etc (Palonen et al., 2003). In present study maximum inhibition was reported on addition of Zn2+ and Mn2+ followed by Fe2+. Comparable to our result More et al., 2011 also reported nearly 60% inhibition of laccase activity in presence of Mn2+ and Fe2+. However metals ions such as Cu2+, Mg2+, K+, Ca2+ have no effect on enzyme activity and to be remains stable in the presence of detergents(Tween 20 and Triton X). More et al., 2011; also reported stability of laccase enzyme in presence of Cu2+, Mg2+ and detergents (Sharma et al., 2013). Decrease in enzyme activity due to detergents is based on the fact that due to surfactants the adsorptions of substrate to the enzyme get reduced. Stability of enzyme in presence of metal ions and surfactants make it a robust enzyme for various biotechnological applications. However in presence of veratryl alcohol no activity was detected enzyme gets completely inhibited. Our results are comparable with the finding of Murugesan et al., 2006. They also reported no for partially purified laccase from Pleurotus sajor-caju. Under standard assay conditions apparent Km and Vmax were calculated 38.5mM and 0.25mM for guaicol. More et al., 2011 reported Km and Vmax of partially purified laccase from Pleurotus sp were 0.25mM and 0.33µmol/min for ABTS respectively. Patel et al., 2014 reported 100mM and 400 mM Km value of laccase from Pleurotus ostreatus HP-1 for guaicol and DMP respectively.

Determination of optimum pH for partially purified laccase activity
The partially purified laccase from G. gibbosum was tested under different pH. The pH showed the maximum activity at pH 5.0 when using guaicol as substrate. Generally fungal laccase showed highest activity between the pH range of 3.0, 5.0 (Shiba et al., 2000; Korolera et al. 2001; Shen et al., 2000). The results obtained are comparable with that of Junghanns et al., 2009, who reported optimum pH values of 2.5, 4.5 and 5.0 for ABTS, DMP and guaicol of purified laccase from Phoma sp. 5-1-03. With increase in pH, a gradual decrease in oxidation rate of various substrates was observed which may be related to ionization of critical amino acids such as Asp and Glu (Salomi et al., 2006), or which may be due to binding of hydroxide ions at T2and T3 site of enzyme (Morozova et al., 2007). Palmieri et al., 1993 reported that Ph optima also varies among the substrates used for eg P. ostreatus has a pH range of 3 – 3.5 for ABTS, 5.6 and 6.7 for guaicol and syringaldazine respectively. Lisova et al. 2010 reported a Ph of 3.8 to be optimum for Cerrena unicolor VKMF-3196 Lac C2 for DMP. Park et al., 2014 reported Ph of 5.5 to be optimum when using guaicol which is also quite comparable with our results. Generally the Ph range for most of the fungal laccase ranges between 3.0 to 5.7, but there are some exceptions which exhibit neutral Ph for various substrates such as Rhizoctonia practicola (Park et al., 2007). Similarly Manimozhi et al., 2012 also reported a Ph value of 5.5 to be optimum for laccase enzyme from Agaricus heterocystis.
Ph Stability
The Ph stability of G. gibbosum laccase is illustrated in Fig. More than 75% and 60% of laccase activity was retained at pH 6.0 and 7.0 respectively at 25°C. Fernaud et al. (2006) reported more than 70% residual activity of Fusarium proliferatum laccase at pH range of 2-9 at 25°C after 2h of incubation. However, Huang et al., 2011 reported more than 95% residual activity of recombinant laccase in pH range of 3.0 – 7.0 at 25°C after 24 h of incubation. When effect of pH on the enzyme stability was examined at 25°C for 1h the enzyme remained stable from 4.0 to 6.0. Our results are comparable with results of Lihua et al., 2009 reported more than 80% activity of Pleurotus ostreatus 10969 at pH value 4.0 after 1h incubation at 25°C. Kumar et al., 2016 reported 91.4% of its initial activity of Aspergillus flavus after 1h and retained 86% of its activity even after 4h of incubation at pH value of 5.0. Park et al., 2007 also reported enzyme from Fomitella fraxinea remained stable within pH range of 3.0 to 5.0 at 25°C for 1h (Park et al., 2007).
Temperature maxima for laccase activity
Optimal temperature range for most of the fungal laccases generally varies between 30°C -60°C (Nishizawa et al., 1995; Wood et al., 1980; Youn et al., 1995). More et al. 2011 reported optimum temperature for laccase from Pleurotus sp was between 30 – 65°C similar to the results of Ryan et al 2003 for laccase from Sclerotium rolfsii. Laccase examined in our study had an optimal temperature range of 25 – 30°C which is similar to that of values obtained for laccase from Ganoderma lucidum (Leem et al. 2001). Fungal laccases have been described as thermostable however most of the white rot fungi laccases are not active beyond a temperature of 50°C. In our study partially purified laccase from G. gibbosum was found to be stable up to 40°C for 3h. However at 50°C considerable loss in laccase activity was recorded (Fig). Stability in enzyme activity at 40°C is probably due to unfolding of protein structure (Ryan et al 2003). In the present study, in the initial 3 hours of incubation laccase activity was found to be stable at 40°C, whereas rapid inactivation of laccase was observed at temperature above 40°C (Fig). Similarly enzyme inactivation at higher temperature was also reported by Agaricus blazei (Ulrich et al. 2005) and Cerrena unicolor LacC2 (Lisova et al., 2010). Park et al. 2007 reported laccase from Fomitella fraxinea remained stable up to 40°C similarly laccase from L. edodes and A. blazei (21,32), however activity decreases rapidly at temperature above 40°C. In contrast laccase enzyme from P. ribis and Trametes sp remains stable at 55 and 70°C for more than one hour as reported by (Min et al.,2001; Xiao et al., 2003). Similarly Tong et al. Gonzalez et al., 2008 also reported stability of laccase from Pycnoporous sanguineusremain stable up to 70°C for 1h. Differences in the effect of incubation temperature on enzyme stability might be related with the number of molecules in the disulphide bonds, thermal dissociation of bonds also participate in temperature profile (Xu et al., 1996).