Effect of veratryl alcohol on ligninase production was tested ranging from 0

Effect of veratryl alcohol on ligninase production was tested ranging from 0.1 to 0.5%. Laccase activity in both fungi increases many folds due to supplementation of production media with VA at 0.4%. Further for a particular fungus-substrate combination with all concentrations of VA, the quantum increase in laccase activity were recorded, amounting 2.1, 5.8, 1.4 and 2.96 fold increase for GWB, LWB,GSD and LSD respectively over their control conditions. LiP and MnP activities were very low, in comparision with laccase in both fungi. Supplementation of VA did not show any appreciable quantitative change in their activity. LiP activity was found to increase only in L. sajor – caju on wheat bran where 1.08 fold increases in LiP activity was recorded, while in case of MnP except L. sajor-caju on wheat bran MnP activity increases slightly amounting 1.2, 1.0 and 1.1 fold for GWB, GSD and LSD respectively. Veratryl alcohol is a compound that either act as a charge transfer mediator (Harvey et al., 1986), a stabilizer (Cancel et al., 1993), an inducer (Faison et al., 1986), a substrate for H2O2-producing enzymes or a source of ROS (De Jong et al., 1994). Even mixed effects cannot be excluded. Even mixed effects cannot be excluded. Jaouani et al 2006 reported stimulatory effect of VA on ligninolytic enzyme from C. polyzona in the range of 5 – 20 mM. Similarly Dekker and Barbosa 2001 also reported stimulatory effect of ligninolytic enzyme from ascomycete strain Botryosphaeria sp. The addition of veratryl alcohol was also shown to enhance laccase activity in growing cultures of numerous white rot fungi as for example Pycnoporus cinnabarinus (Eggert et al., 1996) and Nematoloma forwardii (Hofrichter and Fritsche, 1997). Hence, it seems necessary that further investigations at molecular level be undertaken to elucidate the concentration effect of veratryl alcohol on the production of the different ligninolytic enzymes by C. polyzona.

Effect of copper and manganese ions on ligninase production
Copper is an essential micronutrient for most of living organisms and copper requirement by microorganisms are usually satisfied by low concentration. However copper present in higher concentration is extremely toxic to microbial cells (Labbe et al 1996). Copper atom generally serves as co-factor in the laccase enzyme catalytic core; therefore required in (milli molar) range (Bertrand et al 2013, Patel et al 2009. Galhaup et al 2002; Stajic et al 2006, reported that copper at various concentration stimulates laccase production in T. pubescenes, P. eryngii and P. ostreatus. This might explains the positive effect of copper on enzyme stabilization. Our findings are in accordance with those results. Makela et al., 2013 reported increased laccase activity on supplementation of Cu2+ at 1.5mmol concentration from Phelbia radiata while Levin et al 2008 found11mmol/L as optimal copper ion concentration for laccase production by Trametes trogii. In the present study it was found that both the isolated fungi, G. gibbosum and L. sajor – caju were found to be more efficient laccase producers. Both of them produce much higher level of laccase than LiP and MnP. In both the strains, laccase activity increases several fold due to supplementation of production medium with metal ions (Cu2+and Mn2+). A drastic increase in laccase activity was recorded at all concentrations (0.1 – 0.4mM) of Cu2+ and Mn2+ in stationary phase (Fig). Patel et al 2016, reported stimulatory effect of copper ion on laccase enzyme production. Maximum laccase production was obtained at 0.3mM concentration. Beyond that he also reported decrease in enzyme production; this may be due to the fact that copper at higher concentration is inhibitor of fungal growth. Stimulatory effect of copper ion is also reported by Xin and Geng 2011 for laccase production from Trametes versicolor. Songulashvili et al., 2011 also reported inducing effect of Cu+2 for enhancing laccase production up to 2 fold by Ganoderma lucidum 447, Similarly Manavalan et al 2013 and Mann et al 2015 also reported positive effect of Cu+2 on laccase production in Cerrena consors and Ganoderma lucidum respectively. In present study Cu2+ ion at 0.2mM was found as the best concentration for G. gibbosum on wheat bran and saw dust and L. sajor – caju on wheat bran providing 28910, 19893 and 31900 IU/g respectively and for L sajor – caju on saw dust Cu2+ at 0.4 mM turns to be better concentration resulting in 34047 IU/g laccase activity, amounting 2.13, 1.7, 5.72 and 6.7- fold increase over control conditions respectively. Manavalan et al 2013 reported 0.4 mM copper sulphate as optimal concentration for inducing laccase production from G. lucidum while Fonseca et al 0.5 Mm copper sulphate for stimulating laccase production from Ganoderma applanatum. Different studies have shown that laccase activity has regulated by copper ions through gene expression induction or translational or post translational regulation (Fonseca et al 2010). After addition of Cu2+, it was found that laccase activity increases more in case of L. sajor – caju and slightly less in G. gibbosum on both the substrates. LiP and MnP activities were very low, in comparision with laccase in both fungi under control conditions and supplementation of Cu2+ did not show any enhancing effect on LiP production except in case of L. sajor- caju on wheat bran 1.2 fold increase in LiP activity was recorded at 0.1 mM Cu2+. Similarly supplementation of Cu2+ did not show any appreciable amount of increase in MnP activity, around 1.05, 1.2, 1.1 fold increase at 0.1mM concentration was recorded in case of L sajor- caju on wheat bran; saw dust and G. gibbosum on saw dust respectively. The results obtained in the study proved that for laccase production for both strains wheat bran turns to be better substrate compared to saw dust. In present study we found stimulatory effect of Mn2+ on laccase activity but did not have any stimulatory effect on MnP activity and LiP activity was found to be repressed in its presence. Our results are in accordance with previous studies by Jaouni et al 2006 they also reported repression of C. polyzona LiP with no significant MnP. Contradictory to our results Bonnarme and Jefferies (1990) reported that Mn2+ plays important role in production of LiP and MnP in different white rot fungi. The addition of Mn2+ resulted in a total repression of C. polyzona LiP with no significant effect on MnP or laccase. Moreover, the presence of Mn2+ was reported to be essential for the secretion of active MnP through a post-transcriptional mode (Manubens et al., 2003).