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Effect of incubation time on lignocellulolytic enzyme production
Time of fermentation has an important impact on the product formation. Enzyme production is related to time of incubation (Gautham et al 2011). In the present study time of incubation for biosynthesis of lignocellulolytic enzyme production by two white rot fungi (Ganoderma gibbosum and Lentinus sajor-caju), shows an interesting pattern of enzyme production in the result section. The time courses of enzyme production were measured for 12 days of production under SSF. Data obtained in this research work, shows an overall trend consisting of decrease in enzyme after a specific time. Laccase activity by G. gibbosum and L. sajor – caju on wheat bran tends to decrease after 6th day of fermentation and both strains on saw dust increases up to day 7th and 10th day of fermentation respectively. In case of LiP activity production G. gibbosum on wheat bran and saw dust increases up to 7th and 9th day respectively and then started to decline thereafter. MnP activity was also found to be maximum between 6th – 10th days of production. Generally most of the basidiomycete’s species are capable of producing simultaneously both hydrolytic and oxidative enzymes. In general white rot fungi are selective lignin degraders and shows high titers of oxidative enzyme production than cellulose. In this way they started to degrade the lignin first to assess cellulose. During the study it was found that lignocellulolytic enzyme activity decreases at different time during production under SSF. This decrease in enzyme activity may be related to several factors that affects enzyme production like, efficiency of extraction method adopted, change in composition of substrate due to formation of some new compounds which will affect or inhibit the enzyme activity; also by repression exerted by product generated due to enzymatic reactions; formation of several iso-enzyme at different time and under different substrate conditions leading to variations in enzyme activity (Mata et al., 2005). Time course for production of cellulase enzyme was investigated and fermented for a period of 3 to 12 days was carried out. Enzyme activity was investigated after interval of 24 h. The result showed that maximum cellulase and hemicellulase were observed at incubation time between 5th to 7th days for both strains on both substrates. Decreases in enzyme activity were recorded with further incubation (Fig). This may be related with factors that discussed earlier as with time macro and micronutrient in the production medium will deplete that will affect the fungal physiology which will leads to inactivation of secretory machinery of enzyme (Rajendran et al., 2013). The result of incubation time for both the strains was found to be comparable with the results of Karthikeyan et al., 2010 and Bairagi., 2016 they also reported 5th day to be optimum incubation time for the production of cellulase from Trichoderma atroviride. Patel et al.2009 reported that due to genetic variations among strains and nature of compositions of substrates, at different time periods maximum ligninolytic enzyme productions were reported among different white rot fungi. For eg a white rot fungi (Datronia sp KAPI0039) was reported by Vaithanomsat et al. 2010 to produce maximum laccase and MnP after 4th and 8th day of cultivation respectively. Sharma et al., 2014 reported 7th day as optimum for laccase production from Ganoderma sp.rckk-02; Fortina et al., 1996 reported that Botrytis cinerea produce appreciable amount of laccase between 5th – 7th days. Laccase from Arthrospira maxima (SAE-25780) was reported to be between 2nd -10th day of fermentation by Scheel et al., 2000; Afreen et al 2016).
Effect of ph on lignocellulolytic enzyme production
The pH of SSF medium had a significant influence on enzyme synthesis, as fungi are very sensitive to variation in pH. Enzyme production depends greatly on pH of the medium, as it influence several enzymatic processes by altering permeability of the cell membrane, availability of metals such as Mg+2, Zn+2, Ca+2, Fe+3 (Meichichi et al 2006). The pH of the production medium was varied from 3.0 to 5.5 for both strains and on both substrates tested for all lignocellulolytic enzymes. However the optimal activity for both strains was obtained at pH of 5.0. Further rise in pH did not show increase in activity, this may be due to poor mycelial growth at higher Ph which interferes with the production. Our results are comparable with the results reported by Ravikumar et al 2012, who also reported maximum laccase activity at Ph 5.0. Similarly Chhaya and Gupte (2013) also reported maximum laccase activity at Ph 5.0 using OFAT approach under solid state fermentation. Decrease in enzyme activity is generally attributed to the fact that due to change in the Ph value which may alter the three dimensional structure of enzyme. Elasayed et al 2012 reported Ph ranges between 4.0 to 5.0 to be suitable for ligninolytic enzyme production from most of the basidiomycetes. Shin and Lee, 2000; Radha et al 2005;Yamanaka et al 2008 also reported ph between 3.0 to 6.0 to be optimum for higher activities of ligninolytic enzymes However there are some exceptions such as Ph 3.0 was reported to be optimum for laccase enzyme production from Trametes trogii TEM H2 by Koeyigi et al. 2012. In the present study maximum cellulase production were obtained at pH 5.0 at the optimum temperature. Reduction in enzyme activities after 5.0. These results are in agreement with the findings of Jaradat et al., 2008) who reported that cellulase enzyme from the active isolate J2 was active over a pH range of 4.0 to 7.0 (Bairagi et al 2016).

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