Partial purification and characterization of enzymes from Pleurotus ostreatus and Lentinus edodes using water hyacinth as a substrate

Sibanda, Nompumelelo (2018)

Thesis

Water hyacinth (Eichhornia crassipes) biomass is a potential raw material as a lignocellulosic feed stock to several applications. However, the chemical structure of the lignocellulosic biomass has to be broken down first to obtain fermentable sugars. White-rot fungi such as Pleurotus ostreatus and Lentinus edodesare known to degrade plant biomass by secreting extracellular enzymes of numerous biotechnological applications. The extracellular enzymes include peroxidase, laccases, cellulases, xylanases and pectinases and they have wide and varied applications in bio-fuels, food, chemical, medical, animal feed, textile and laundry, pulp and paper industries. The versatile nature of P. ostreatus and L. edodes provides a basis for exploration of biochemical processes during solid state fermentation (SSF) on E. crassipes(water hyacinth) as a lignocellulosic biomass. White-rot fungi degrade substrate by secreting extracellular enzymes, which break down the lignocellulose polymer into smaller compounds that the hyphae then absorb and utilize for growth and development. The objectives of the study were to qualitatively profile, isolate, partially purify and characterize enzymes from P. ostreatus and L. edodes grown on water hyacinth as a substrate. The last objective was to purify the extracted enzyme using different purification agents as follows: ammonium sulfate, acetone and activated carbon. The fungi were cultured separately on E. crassipes using the solid state fermentation method under controlled conditions and mycelium development was monitored. At full colonization, enzyme extraction was done and crude enzyme filtrate on Agar Plate-based Clearing Assays showed the presence of extracellular enzymes, namely cellulases, xylanases, pectinases, lignin peroxidases, and laccases which were further consolidated by spectrophotometric activity assays. The Michaelis constant (Km) of laccase was lower at 0.145 mM from P. ostreatus while for cellulase it was 0.246 mM. The Km of cellulase from L. edodes was at 0.248 mM. Partial purification of cellulases and laccases with ammonium sulfate, acetone or activated charcoal showed activated charcoal to be more effective and efficient compared to ammonium sulfate and acetone as shown by a marked increase in enzyme activity and clarification of the enzyme solution after purification. An SDS-PAGE analysis of the enzyme fractions secreted by P. ostreatus on E. crassipes revealed molecular mass of 85 kDa for laccases and 50 kDa for cellulases while a zymogram analysis of the two enzymes demonstrated activity in the corresponding ranges of 40 and 50 kDa for cellulases and between 60 to 85 kDa for laccase. This study showed the ability of P.ostreatus and L.edodesto produce fungal enzymes and convert the biomass into valuable substances during biodegradation of E. crassipes

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