Screening diverse cellulase enzymes from the white rot fungus Phlebia gigantea for high activity and large scale applications
Creators: name: Niranjane, Ajay
Publisher: name: RMIT University
Year: 2024

description: Cellulosic biomass is the major organic matter produced in the biosphere. The biodegradation of this cellulosic material is achieved by enzymatic activities of the cellulose degrading microorganisms. These organisms usually express a complex extracellular or a membrane bound cellulolytic system comprising combination of several cellulase enzymes. Cellulases are the group of hydrolytic enzymes capable of hydrolysing insoluble cellulose to glucose. <br><br>Phlebia gigantea is an aggressive white rot basidiomycete with ability to tolerate resinous extracts on freshly cut wood and higher growth rate. This helps the fungus to colonise the sapwood preventing other fungi from becoming established. Early research on the cellulase system of this organism reported the presence of a cellulase system composed of β-glucosidase, endoglucanase and a cellobiohydrolase. Based on these unpublished studies, our aim was to obtain a complete sequence of putative cellobiohydrolase I (CbhI) from this organism. Attempts to identify and isolate the cellulase gene resulted in an incomplete cDNA sequence of 1154 bp. <br><br>To understand the cellulase system, expression and regulation of the cellulase enzymatic activity was examined for incubation of P. gigantea on substrates glucose, xylose, Avicel, carboxymethyl cellulose and cellobiose. The pH, total protein and biomass production results indicated that the capacity of P. gigantea to degrade cellulose is dependent upon the nature of the carbon source and the regulation of the cellulase synthesis is repressed in the presence of simple sugars like glucose and xylose. <br><br>The study employed the highly effective method of purification by affinity adsorption and purified cellulase complex in large quantity. Characterisation of the kinetic properties of this cellulase complex revealed that the rate of cellulase catalysis were optimum at pH 5.0 and temperature 50GC. The purified complex was comprised of multiple proteins and demonstrated significant CMCase and CBHase activity on zymogram analysis. The purified cellulase complex was characterised by 2D gel electrophoresis and by peptide mass finger printing using MALDI-TOF massspectrometry analysis. <br><br>The 2D gel analysis of the purified cellulase complex showed 15 spots within the range of pI 3.5 to pI 7 and the molecular weight between 20KDa to 100KDa. Three protein spots were selected based on the IEF and SDS zymogram and identified using MALDI-TOF MS analysis. These proteins were identified based on the peptide mass data belonging to the 6-phospho-α-glucosidase, β-glucosidase and glycosyl hydrolase family 13 a-amylase or pullulanases, suggesting the divergent evolution of specific cellulase proteins. This study showed P. gigantea as a potential cellulase source and the cellulase complex secreted by the induction of substrate, comprises a variety of enzymes related to hydrolysis of cellulose biomass. It is evident from this and previous studies that P. gigantea cellulase complex comprises of a specific set of enzymes that possess the ability to degrade crystalline cellulose and is one of the first organisms to colonise freshly cut wood. Further studies on the cellulase system of this primary colonist may open up the prospects to utilise this organism as the potential onsite bioreactor agent, pre-treating the biomass and increasing the economic feasibility of the industrial bioenergy processes.<br>